Cation complexes of insulin compound conjugates, formulations and uses thereof

ABSTRACT

An insulin compound coupled to a modifying moiety having a formula: 
       —X—R 1 —Y-PAG-Z—R 2    (Formula VI)
         where,   X, Y and Z are independently selected linking groups and each is optionally present, and X, when present, is coupled to the insulin compound by a covalent bond,   either R 1  or R 2  is is a lower alkyl, optionally including a carbonyl group, and when R 1  is a lower alkyl, R 2  is a capping group, and   PAG is a linear or branched carbon chain incorporating one or more alkalene glycol moieties, and optionally incorporating one or more additional moieties selected from the group consisting of —S—, —O—, —N—, and —C(O)—.

1 RELATED APPLICATIONS

This application is a divisional application of and claims priority toco-pending U.S. patent application Ser. No. 11/184,668, now U.S. Pat.No. 7,875,700, which in turn claims claims priority to and incorporatesby reference the entire disclosures of U.S. Patent Application Nos.60/589,058, filed Jul. 19, 2004; 60/619,153, filed Oct. 15, 2004;60/632,578, filed Dec. 2, 200; 60/655,838, filed Feb. 24, 2005; and60/655,803, filed Feb. 24, 2005. which in turn claims priority to andincorporates by reference the entire disclosures of U.S. PatentApplication Nos. 60/589,058, filed Jul. 19, 2004; 60/619,153, filed Oct.15, 2004; 60/632,578, filed Dec. 2, 200; 60/655,838, filed Feb. 24,2005; and 60/655,803, filed Feb. 24, 2005. This application alsoincorporates by reference the following applications filed herewith onJul. 19, 2005 by Radhakrishnan et al.: U.S. patent application Ser. No.11/184,594, entitled “INSULIN-OLIGOMER CONJUGATES, FORMULATIONS AND USESTHEREOF,” now U.S. Pat. No. 7,872,095; U.S. patent application Ser. No.11/184,528, entitled “FATTY ACID FORMULATIONS FOR ORAL DELIVERY OFPROTEINS AND PEPTIDES, AND USES THEREOF,” now U.S. Pat. No. 7,605,123;International Patent Application No. PCT/US05/25644, entitled “INSULINCOMPOUND CONJUGATES, CATION COMPLEXES, FORMULATIONS AND USES THEREOF.”

2 FIELD

The invention relates generally to complexes in which a metal cation iscomplexed with an insulin compound conjugate, and to the making andusing of such complexes. The complexes may be provided as solids in aliquid or as dried solids, and may be provided as components ofpharmaceutical formulations. The complexes exhibit various propertiesuseful in the preparation of pharmaceuticals and in the treatment ofinsulin compound deficiencies. The invention also relates to novel novelmodifying moieties for making drug conjugates and to novel proteinconjugates, including insulin compound conjugates, employing suchmodifying moieties. Further, the invention relates to novel fatty acidformulations and to such formulations prepared for oral delivery ofconjugated and unconjugated proteins and peptides, such as the insulincompound conjugates and complexes of the invention.

3 BACKGROUND

Zinc complexed insulin compound is commercially available, for example,under the trade names HUMULIN® and HUMALOG®. Zinc complexed insulintypically exists in a hexameric form.

Various methods have been described for the use of zinc in thecrystallization of acylated insulin. For example, U.S. PatentPublication 20010041786, published on 15 Nov. 2001, by Mark L. Brader etal., entitled “Stabilized acylated insulin formulations” describes aformulation with an aqueous solution for parenteral delivery,particularly as an injectable formulation, with a pH of 7.1 to 7.6,containing a fatty acid-acylated insulin or a fatty acid-acylatedinsulin analog and stabilized using zinc and preferably a phenoliccompound. U.S. Pat. No. 6,451,970, issued on 17 Sep. 2002 to Schaffer etal., assigned to Novo Nordisk AIS, entitled “Peptide derivatives”describes derivatives of insulin compound and insulin analogs where theN-terminal amino group of the B-chain and/or the ε-amino group of Lys inposition B28, B29 or B30 is acylated using long chain hydrocarbon grouphaving from 12 to 22 carbon atoms and zinc complexes thereof.

Protamines and phenolic compounds have been described for use in thecrystallization of acylated insulin. U.S. Pat. No. 6,268,335 (31 Jul.2001) and U.S. Pat. No. 6,465,426 (10 Oct. 2002) to Brader, bothentitled “Insoluble insulin compositions,” describe insolublecompositions comprised of acylated insulin a protamine complexingcompound, a hexamer-stabilizing phenolic compound, and a divalent metalcation.

Existing approaches are especially tailored for crystallization ofnative insulin compound or insulin compound analogs or for acylatedinsulin compounds having increased lipophilicity relative tonon-acylated insulin compounds. There is a need in the art forpharmaceutically acceptable complexes including derivatized insulincompounds, other than acylated insulin compound, such as hydrophilicand/or amphiphilic insulin compound derivatives, and for stabilizingnon-acylated lipophilic insulin compound analogs. There is also a needin the art for new protein conjugates having increased bioavailabilityor other improved pharmaceutical attributes relative to existingconjugates. There is a need in the art for new formulations thatfacilitate oral delivery of proteins and protein conjugates. Finally,there is a need for a combined approach to improving the oralbioavailability of a protein, such as insulin compound, whichincorporates an improved oral protein conjugate provided as a solid inan improved formulation to maximize the benefits for the oral deliveryof proteins.

4 SUMMARY OF THE INVENTION

In general, the invention provides a complex including an insulincompound conjugate with an insulin compound conjugated to a modifyingmoiety, and a cation, where the insulin compound conjugate is complexedwith the cation. The insulin compound may, for example, be a nativeinsulin or an insulin analogs. Examples of insulin compounds includehuman insulin, lyspro insulin, des30 insulin, native proinsulin,artificial proinsulins, etc. The cation component may, for example, be adivalent metal cation selected from the group consisting of Zn++, Mn++,Ca++, Fe++, Ni++, Cu++, Co++ and Mg++.

The modifying moiety may be selected to render the insulin compoundconjugate more, less or equally soluble as compared to the correspondingunconjugated insulin compound. The modifying moiety is preferablyselected to render the insulin compound conjugate at least 1.05, 1.25,1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 times moresoluble than a corresponding unconjugated insulin compound in an aqueoussolution at a pH of about 7.4. Preferably the modifying moiety isselected to render an insulin compound conjugate having an aqueoussolubility that exceeds about 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 10 g/L,15 g/L, 20 g/L, 25 g/L, 50 g/L, 75 g/L, 100 g/L, 125 g/L, or 150 g/L ata pH of about 7.4. Further, the the modifying moiety is selected torender the insulin compound conjugate equally or more soluble than acorresponding unconjugated insulin compound, and the water solubility ofthe insulin compound conjugate is decreased by the addition of zinc. Inanother embodiment, the modifying moiety is selected to render theinsulin compound conjugate equally or more soluble than a correspondingunconjugated insulin compound; the water solubility of the insulincompound conjugate is decreased by the addition of zinc; and watersolubility of the complex is greater than the water solubility ofinsulin compound. In still another embodiment, the relativelipophilicity of the insulin compound conjugate as compared tocorresponding parent insulin compound (k_(rel)) is 1 or less than 1.

The invention also provides novel insulin compound conjugates having aninsulin compound conjugated to a modifying moiety. For example, theinvention provides insulin compounds coupled to a modifying moietyhaving a formula:

—X—R¹—Y-PAG-Z—R²   (Formula VI)

where,

X, Y and Z are independently selected linking groups and each isoptionally present, and X, when present, is coupled to the insulincompound by a covalent bond,

at least one of R¹ and R² is present, and is lower alkyl and mayoptionally include a carbonyl group,

R² is a capping group, such as —CH₃, —H, tosylate, or an activatinggroup, and

PAG is a linear or branched carbon chain incorporating one or morealkalene glycol moieties (i.e., oxyalkalene moieties), and optionallyincorporating one or more additional moieties selected from the groupconsisting of —S—, —O—, —N—, and —C(O)—, and

where the modifying moiety has a maximum number of 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 heavyatoms.

In embodiments of the invention, any one or more of X, Y and Z may beabsent. Further, when present, X, Y and/or Z may be independentlyselected from —C(O)—, —O—, —S—, —C— and —N—. In one embodiment, Z is—C(O)—. In another embodiment, Z is not present.

In some embodiments, R¹ is lower alkyl, and R² is not present. In otherembodiments, R² is lower alkyl, and R¹ is not present.

In another embodiment, the modifying moiety may include a linear orbranched, substituted carbon chain moiety having a backbone of 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24or 25 atoms selected from the group consisting of —C, —C—, —O—, ═O, —S—,—N—, —Si—. The heavy atoms will typically include one or more carbonatoms and one or more non-carbon heavy atoms selected from the groupconsisting of —O—, —S—, —N—, and ═O. The carbon atoms and non-carbonheavy atoms are typically present in a ratio of at least 1 carbon atomfor every non-carbon heavy atom, preferably at least 2 carbon atoms forevery non-carbon heavy atom, more preferably at least 3 carbon atoms forevery non-carbon heavy atom. The carbon atoms and oxygen atoms aretypically present in a ratio of at least 1 carbon atom for every oxygenatom, preferably at least 2 carbon atoms for every oxygen atom, morepreferably at least 3 carbon atoms for every oxygen atom. The modifyingmoiety may include one or more capping groups, such as branched orlinear C₁₋₆, branched or linear, or a carbonyl. The modifying moietywill typically include hydrogens, and one or more of the hydrogens maybe substituted with a fluorine (which is a heavy atom but should not becounted as a heavy atom in the foregoing formula). The modifying moietymay in some cases specifically exclude unsubstituted alkyl moieties. Themodifying moiety may, for example, be coupled to an available group onan amino acid, such as an amino group, a hydroxyl group or a freecarboxylic acid group the polypeptide, e.g., by a linking group, such asa carbamate, carbonate, ether, ester, amide, or secondary amine group,or by a disulfide bond. The molecules in the linking group are countedas part of the modifying moiety. In a preferred embodiment, themolecular weight of the modifying moiety is less than the molecularweight of the HIM2 modifying moiety.

The invention includes includes insulin compound conjugates havingmodifying moieties with a formula:

where n is 1, 2, 3 or 4, and m is 1, 2, 3, 4 or 5; and/or

where n is 1, 2, 3, 4 or 5, and m is 1, 2, 3 or 4.

It will be appreciated that the novel modifying moieties, as well as theuse of such moieties to modify insulin and other polypeptides arethemselves aspects of the invention.

The invention also provides novel formulations including the insulincompound conjugates and/or cation-insulin compound conjugates of theinvention. The inventors have surprisingly discovered that certain fattyacid compositions are particularly useful, especially for oral deliveryof the polypeptides and polypeptide conjugates, such as insulin andinsulin compound conjugates and/or oral delivery of the cation-insulincompound conjugate complexes of the invention. In one aspect, theinvention provides fatty acid compositions with one or more saturated orunsaturated C₄, C₅, C₆, C₇, C₈, C₉ or C₁₀ fatty acids and/or salts ofsuch fatty acids. Preferred fatty acids are caprylic, capric, myristicand lauric. Preferred fatty acid salts are sodium salts of caprylic,capric, myristic and lauric acid. The fatty acid content of thecomposition is typically within a range having as a lower limit of about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, or 3.0% w/w, and having as an upper limit of about 3.0, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8,8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2,10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4,11.5, 11.6, 11.7, 11.8, 11.9, or 12.0% w/w. In yet another embodiment,the fatty acid content of the composition is within a range having as alower limit about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, or 3.0% w/w, and having as an upper limit about 3.0,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0,10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2,11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, or 12.0% w/w, and the fattyacid content of the composition is typically greater than about 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, or 99.9% w/w asingle fatty acid, preferably caprylic, capric, myristic or lauric, or asalt thereof.

The invention also provides method of treating insulin deficiencies orotherwise supplementing insulin in a subject using the insulin compoundconjugates, cation-insulin compound conjugate complexes, and/orformulations of the invention. The methods generally includeadministering a therapeutically effective amount of one or more of thethe insulin compound conjugates, cation-insulin compound conjugatecomplexes, and/or formulations of the invention to a subject in needthereof.

5 BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-15B show photomicrographs of various crystalline solids of theinvention. FIGS. 1 and 2 are photomicrographs taken using a ZeissAxiovert microscope showing T-type Zn complex of of HIM2 30 g/Lconcentration, crystals grown for 24 hours. FIG. 3 is a photomicrographtaken using a Zeiss Axiovert microscope showing T-type Zn complex of ofHIM2 30 g/L concentration, crystals grown for 5 days. FIG. 4 is aphotomicrograph taken using a Zeiss Axiovert microscope showing R-typeZn complex of HIM2 at 30 g/L crystals grown for 4 days. FIG. 5 showsphotomicrograph of R-type crystalline Zn complex of IN105 containing 30%organic. FIGS. 6A-10B show photomicrographs of various R-type Zncomplexes of HIM2 made using organic solvent. FIGS. 11A-14B showphotomicrographs of crystals of various R-type co-crystallized Zncomplexes of HIM2 and IN105. FIGS. 15A-15B show photomicrographs ofcrystals of various R-type co-crystallized Zn complexes of HIM2 andhuman insulin. The invention includes crystals having the morphologiesshown in any of FIGS. 1-15B.

FIGS. 16-20 show Mouse Blood Glucose Assay results for HIM2 and variousZn-HIM2 complexes. FIG. 16 shows MBGA biopotency profiles for HIM2. FIG.17 shows MBGA biopotency profiles for Zn HIM2 insulin compound product Rtype. FIG. 18 shows MBGA biopotency profiles for Zn HIM2 insulincompound product T-type. FIG. 19 shows MBGA biopotency profiles for ZnHIM2 insulin compound product with protamine. FIG. 20 shows glucoselowering effect of R type protamine complex at 30 and 90 minutes postdose.

FIGS. 21-24 show MBGA biopotency profiles for IN-186, IN-192, IN-190,IN-191, IN-189, IN-178, IN-193, IN-194, IN-185, IN-196 and IN-197.

FIGS. 25 and 26 show dog clamp study results for Zn-HIM2 complexes ofthe invention. FIGS. 27 and 28 show dog clamp study results for Zn-IN105complexes of the invention.

FIGS. 29 and 30 show dog clamp study results for dogs dosed with IN105in 3% w/v capric acid sodium salt in a phosphate buffer withoutadditional excipients.

FIGS. 31-33 show dog clamp study results for dogs dosed with tabletscontaining 6 mg of IN105 and 150 mg Mannitol, 30 mg Exlotab with 143 mgCaparte with or with out 143 mg laurate.

FIGS. 34-37 show dog clamp study results for dogs dosed with prototypetablet 150 mg and 280 mg caprate tablets and with 140 mg/140 mgcaprate/laurate tablets.

FIGS. 38-42 show dog clamp study results for additional dogs dosed withprototype tablet 150 mg and 280 mg caprate tablets and with 140 mg/140mg caprate/laurate tablets.

6 DEFINITIONS

The following are definitions of the terms as used throughout thisspecification and claims. The definitions provided apply throughout thepresent specification unless otherwise indicated. Terms not definedherein have the meaning commonly understood in the art to which the termpertains.

“Addition,” when used in reference to an amino acid sequence, includesextensions of one or more amino acids at either or both ends of thesequence as well as insertions within the sequence.

“Complex” refers to a molecular association in which one or more insulincompounds or insulin compound conjugates form coordinate bonds with oneor more metal atoms or ions. Complexes may exist in solution or as asolid, such as a crystal, microcrystal, or an amorphous solid.

“Complex mixture” means a mixture having two or more differentcomplexes, whether in solution or in solid form. Complexes mixtures may,for example, include complexes with different insulin compounds,different insulin compound conjugates, different hybrid complexes,different cations, combinations of the foregoing, and the like. “Hybridcomplex” means a cation-insulin compound conjugate complex having two ormore different insulin compounds and/or insulin compound conjugates.

“Complexing agent” means a molecule that has a multiplicity of chargesand that binds to or complexes with insulin compound conjugates.Examples of complexing agents suitable for use in the present inventioninclude protamines, surfen, globin proteins, spermine, spermidinealbumin, amino acids, carboxylic acids, polycationic polymer compounds,cationic polypeptides, anionic polypeptides, nucleotides, and antisense.See Brange, J., Galenics of Insulin compound, Springer-Verlag, BerlinHeidelberg (1987), the entire disclosure of which is incorporated hereinby reference.

“Conservative” used in reference to an addition, deletion orsubstitution of an amino acid means an addition, deletion orsubstitution in an amino acid chain that does not completely diminishthe therapeutic efficacy of the insulin compound, i.e., the efficacy maybe reduced, the same, or enhanced, relative to the therapeutic efficacyof scientifically acceptable control, such as a corresponding nativeinsulin compound.

“Hydrophilic” means exhibiting characteristics of water solubility, andthe term “hydrophilic moiety” refers to a moiety which is hydrophilicand/or which when attached to another chemical entity, increases thehydrophilicity of such chemical entity. Examples include, but are notlimited to, sugars and polyalkylene moieties such as polyethyleneglycol. “Lipophilic” means exhibiting characteristics of fat solubility,such as accumulation in fat and fatty tissues, the ability to dissolvein lipids and/or the ability to penetrate, interact with and/or traversebiological membranes, and the term, “lipophilic moiety” means a moietywhich is lipophilic and/or which, when attached to another chemicalentity, increases the lipophilicity of such chemical entity.

“Amphiphilic” means exhibiting characteristics of hydropilicity andlipophilicity, and the term “amphiphilic moiety” means a moiety which isamphiphilic and/or which, when attached to a polypeptide ornon-polypeptide drug, increases the amphiphilicity (i.e., increases boththe hydrophilicity and the amphiphilicity) of the resulting conjugate,e.g., certain PEG-fatty acid modifying moieties, and sugar-fatty acidmodifying moieties.

“Lower alkyl” means substituted or unsubstituted, linear or branchedalkyl moieties having from one to six carbon atoms, i.e., C₁, C₂, C₃,C₄, C₅ or C₆. “Higher alkyl” means substituted or unsubstituted, linearor branched alkyl moieties having six or more carbon atoms, e.g., C₇,C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, etc.

“Monodispersed” describes a mixture of compounds where about 100 percentof the compounds in the mixture have the same molecular weight.“Substantially monodispersed” describes a mixture of compounds where atleast about 95 percent of the compounds in the mixture have the samemolecular weight. “Purely monodispersed” describes a mixture ofcompounds where about 100 percent of the compounds in the mixture havethe same molecular weight and have the same molecular structure. Thus, apurely monodispersed mixture is a monodispersed mixture, but amonodispersed mixture is not necessarily a purely monodispersed mixture.“Substantially purely monodispersed” describes a mixture of compoundswhere at least about 95 percent of the compounds in the mixture have thesame molecular weight and same molecular structure. Thus, asubstantially purely monodispersed mixture is a substantiallymonodispersed mixture, but a substantially monodispersed mixture is notnecessarily a substantially purely monodispersed mixture. The insulincompound conjugate components of the cation-insulin compound conjugatecompositions are preferably monodispersed, substantially monodispersed,purely monodispersed or substantially purely monodispersed, but may alsobe polydispersed. “Polydispersed” means having a dispersity that is notmonodispersed, substantially monodispersed, purely monodispersed orsubstantially purely monodispersed.

“Native insulin compound” as specifically used herein means mammalianinsulin compound (e.g., human insulin, bovine insulin compound, porcineinsulin compound or whale insulin compound), provided by natural,synthetic, or genetically engineered sources. Human insulin is comprisedof a twenty-one amino acid A-chain and a thirty-amino acid B-chain whichare cross-linked by disulfide bonds. A properly cross-linked humaninsulin includes three disulfide bridges: one between A7 and B7, asecond between A20 and B19, and a third between A6 and A11. Humaninsulin possesses three free amino groups: B1-Phenylalanine, A1-Glycine,and B29-Lysine. The free amino groups at positions A1 and B1 are α-aminogroups. The free amino group at position B29 is an ε-amino group.

“Insulin analog” means a polypeptide exhibiting some, all or enhancedactivity relative to a corresponding native insulin or which isconverted in in vivo or in vitro into a polypeptide exhibiting ome, allor enhanced activity relative to a corresponding native insulin, e.g., apolypeptide having the structure of a human insulin with one or moreconservative amino acid additions, deletions and/or substitutions.Insulin analogs can be identified using known techniques, such as thosedescribed in U.S. Patent Publication No. 20030049654, “Protein designautomation for protein libraries,” filed 18 Mar. 2002 in the name ofDahiyat et al. Proinsulins, pre-proinsulins, insulin precursors, singlechain insulin precursors of humans and non-human animals and analogs ofany of the foregoing are also referred to herein as insulin analogs, asare non-mammalian insulins. Many insulin analogs are known in the art(see discussion below). Unless context specifically indicates otherwise(e.g., were a specific insulin is referenced, such as “human insulin” orthe like), the term “insulin compound” is used broadly to include nativeinsulins and insulin analogs.

“Polyalkylene glycol” or PAG refers to substituted or unsubstituted,linear or branched polyalkylene glycol polymers such as polyethyleneglycol (PEG), polypropylene glycol (PPG), and polybutylene glycol (PBG),and combinations thereof (e.g., linear or branched polymers includingcombinations of two or more different PAG subunits, such as two or moredifferent PAG units selected from PEG, PPG, PPG, and PBG subunits), andincludes the monoalkylether of the polyalkylene glycol. The term PAGsubunit means a single PAG unit, e.g., “PEG subunit” refers to a singlepolyethylene glycol unit, e.g., —(CH₂CH₂O)—, “PPG subunit” refers to asingle polypropylene glycol unit, e.g., —(CH₂CH₂CH₂O)—, and “PBGsubunit” refers to a single polypropylene glycol unit, e.g.,—(CH₂CH₂CH₂CH₂O)—. PAGs and/or PAG subunits also include substitutedPAGs or PAG subunits, e.g., PAGs including alkyl side chains, such asmethyl, ethyl or propyl side chains, or carbonyl side chains, as well asPAGs including one or more branched PAG subunits, such as iso-PPG oriso-PBG.

“Proinsulin compound” means an insulin compound in which the C-terminusof the B-chain is coupled to the N-terminus of the A-chain via a naturalor artificial C-peptide having 5 or more amino acids. “Preproinsulincompound” means a proinsulin compound further including a leadersequence coupled to the N-terminus of the B -chain, such as a sequenceselected to promote excretion as a soluble protein, or a sequenceselected to prevent conjugation of the N-terminus, or a sequenceselected to enhance purification (e.g., a sequence with binding affinityto a purification column) “Single chain insulin compound precursor” or“miniproinsulin compound” means an insulin compound in which theC-terminus of the B-chain (or a truncated B-chain having 1, 2, 3 or 4amino acids removed from the C-terminus) is coupled to the N-terminus ofthe A-chain or a truncated A-chain shortened at the N-terminus by 1, 2,3 or 4 amino acids, without an intervening C-peptide, or via a shortenedC-peptide having 1, 2, 3 or 4 amino acids.

“Protamine” refers to a mixture of strongly basic proteins obtained fromnatural (e.g., fish sperm) or recombinant sources. See Hoffmann, J. A.,et al., Protein Expression and Purification, 1:127-133 (1990). TheProtamine composition can be provided in a relatively salt-freepreparation of the proteins, often called “protamine base” or in apreparation including salts of the proteins.

“Protein” “peptide” and “polypeptide” are used interchangeably herein torefer to compounds having amino acid sequences of at least two and up toany length.

“R-type” means a complex conformation formed in the presence of insulincompound conjugate, a cation and a stabilizing compound, such as phenol.“T-type” means a complex conformation formed in the presence of insulincompound conjugate and a cation without a stabilizing compound, such asphenol. A T-type or R-type complex may include or exclude protamine.

“Scientifically acceptable control” means an experimental control thatis acceptable to a person of ordinary skill in the art of the subjectmatter of the experiment.

“Solid” means a state of matter in which there is three-dimensionalregularity of structure; the term is used broadly herein to refer toboth crystalline solids, amorphous solids, and combinations ofcrystalline solids and amorphous solids. “Cation-insulin compoundconjugate solid,” refers to a solid that includes a cation-insulincompound conjugate, preferably coordinated with a monovalent ormultivalent cation. “Crystal” means a solid with a regular polyhedralshape. “Crystalline” refers to solids having the characteristics ofcrystals. “Microcrystal” means a solid that is comprised primarily ofmatter in a crystalline state that is microscopic in size, typically oflongest dimension within the range 1 micron to 100 microns. In somecases, the individual crystals of a microcrystalline composition arepredominantly of a single crystallographic composition. In someembodiments, the crystals of the invention are not microcrystals. Theterm “microcrystalline” refers to the state of being a microcrystal.“Amorphous” refers to a solid material that is not crystalline in form.The person of ordinary skill in the art can distinguish crystals fromamorphous materials using standard techniques, e.g., using x-raycrystallographic techniques, scanning electron microscopy or opticalmicroscopy. “Solid mixture” means a mixture of two different solids.“Crystal mixture” means a mixture of two different crystals.“Co-crystal” means a crystal having two or more different insulincompounds and/or insulin compound conjugates. The cation-insulincompound conjugate complexes of the invention may be provided in any ofthe foregoing forms or in mixtures of two or more of such forms.

“Substitution” means replacement of one or more amino acid residueswithin the insulin compound sequence with another amino acid. In somecases, the substituted amino acid acts as a functional equivalent,resulting in a silent alteration. Substitutions may be conservative; forexample, conservative substitutions may be selected from other membersof the class to which the substituted amino acid belongs. Examples ofnonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. Examples ofpolar neutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine, and glutamine. Examples of positively charged(basic) amino acids include arginine, lysine and histidine. Examples ofnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

“Water solubility” or “aqueous solubility” unless otherwise indicated,is determined in an aqueous buffer solution at a pH of 7.4.

7 DETAILED DESCRIPTION OF THE INVENTION

The invention provides cation-insulin compound conjugate complexes andvarious compositions including such complexes, as well as methods ofmaking and using such complexes and compositions. The complexes areuseful for administering insulin compound for the treatment of variousmedical conditions, such as conditions characterized by insulin compounddeficiency. The complexes generally include a cation component and aninsulin compound conjugate component. The insulin compound conjugatecomponent generally includes an insulin compound coupled to a modifyingmoiety. Examples of other suitable components of the complexes and/orcompositions include stabilizing agents, complexing agents, and othercomponents known in the art for use in preparing cation-proteincomplexes. The invention also provides novel insulin compound conjugatesand fatty acid formulations including such insulin compound conjugatesand/or cation-insulin compound conjugate complexes.

7.1 Insulin Compound

The cation-insulin compound conjugate includes an insulin compoundcomponent. The insulin compound may, for example, be a mammalian insulincompound, such as human insulin, or an insulin compound analog.

A wide variety of insulin compound analogs are known in the art.Preferred insulin compound analogs are those which include a lysine,preferably a lysine within 5 amino acids of the C-terminus of the Bchain, e.g., at position B26, B27, B28, B29 and/or B30. A set ofsuitable analogs is described in EP-A 1227000107 (the entire disclosureof which is incorporated herein by reference), having the sequence ofinsulin compound, except that the amino acid residue at position B28 isAsp, Lys, Leu, Val, or Ala; the amino acid residue at position B29 isLys or Pro; the amino acid residue at position B10 is His or Asp; theamino acid residue at position B1 is Phe, Asp, or deleted alone or incombination with a deletion of the residue at position B2; the aminoacid residue at position B30 is Thr, Ala, or deleted; and the amino acidresidue at position B9 is Ser or Asp; provided that either position B28or B29 is Lys.

Other examples of suitable insulin compound analogs include Asp^(B28)human insulin, Lys^(B28) human insulin, Leu^(B28) human insulin,Val^(B28) human insulin, Ala^(B28) human insulin, Asp^(B28)Pro^(B29)human insulin, Lys^(B28)Pro^(B29) human insulin, Leu^(B28)Pro^(B29)human insulin, Val^(B28)Pro^(B29) human insulin, Ala^(B28)Pro^(B29)human insulin, as well as analogs provided using the substitutionguidelines described above. Insulin compound fragments include, but arenot limited to, B22-B30 human insulin, B23-B30 human insulin, B25-B30human insulin, B26-B30 human insulin, B27-B30 human insulin, B29-B30human insulin, B1-B2 human insulin, B1-B3 human insulin, B1-B4 humaninsulin, B1-B5 human insulin, the A chain of human insulin, and the Bchain of human insulin.

Still other examples of suitable insulin compound analogs can be foundin U.S. Patent Publication No. 20030144181A1, entitled “Insolublecompositions for controlling blood glucose,” 31 Jul. 2003; U.S. PatentPublication No. 20030104983A1, entitled “Stable insulin formulations,” 5Jun. 2003; U.S. Patent Publication No. 20030040601A1, entitled “Methodfor making insulin precursors and insulin analog precursors,” 27 Feb.2003; U.S. Patent Publication No. 20030004096A1, entitled “Zinc-free andlow-zinc insulin preparations having improved stability,” 2 Jan. 2003;U.S. Pat. No. 6,551,992B1, entitled “Stable insulin formulations,” 22Apr. 2003; U.S. Pat. No. 6,534,288B1, entitled “C peptide for improvedpreparation of insulin and insulin analogs,” 18 Mar. 2003; U.S. Pat. No.6,531,448B1, entitled “Insoluble compositions for controlling bloodglucose,” 11 Mar. 2003; U.S. Pat. No. RE37,971E, entitled “Selectiveacylation of epsilon-amino groups,” 28 Jan. 2003; U.S. PatentPublication No. 20020198140A1, entitled “Pulmonary insulin crystals,” 26Dec. 2002; U.S. Pat. No. 6,465,426B2, entitled “Insoluble insulincompositions,” 15 Oct. 2002; U.S. Pat. No. 6,444,641B1, entitled “Fattyacid-acylated insulin analogs,” 3 Sep. 2002; U.S. Patent Publication No.20020137144A1, entitled “Method for making insulin precursors andinsulin precursor analogues having improved fermentation yield inyeast,” 26 Sep. 2002; U.S. Patent Publication No. 20020132760A1,entitled “Stabilized insulin formulations,” 19 Sep. 2002; U.S. PatentPublication No. 20020082199A1, entitled “Insoluble insulincompositions,” 27 Jun. 2002; U.S. Pat. No. 6,335,316B1, entitled “Methodfor administering acylated insulin,” 1 Jan. 2002; U.S. Pat. No.6,268,335B1, entitled “Insoluble insulin compositions,” 31 Jul. 2001;U.S. Patent Publication No. 20010041787A1, entitled “Method for makinginsulin precursors and insulin precursor analogues having improvedfermentation yield in yeast,” 15 Nov. 2001; U.S. Patent Publication No.20010041786A1, entitled “Stabilized acylated insulin formulations,” 15Nov. 2001; U.S. Patent Publication No. 20010039260A1, entitled“Pulmonary insulin crystals,” 8 Nov. 2001; U.S. Patent Publication No.20010036916A1, entitled “Insoluble insulin compositions,” 1 Nov. 2001;U.S. Patent Publication No. 20010007853A1, entitled “Method foradministering monomeric insulin analogs,” 12 Jul. 2001; U.S. Pat. No.6,051,551A, entitled “Method for administering acylated insulin,” 18Apr. 2000; U.S. Pat. No. 6,034,054A, entitled “Stable insulinformulations,” 7 Mar. 2000; U.S. Pat. No. 5,970,973A, entitled “Methodof delivering insulin lispro,” 26 Oct. 1999; U.S. Pat. No. 5,952,297A,entitled “Monomeric insulin analog formulations,” 14 Sep. 1999; U.S.Pat. No. 5,922,675A, entitled “Acylated Insulin Analogs,” 13 Jul. 1999;U.S. Pat. No. 5,888,477A, entitled “Use of monomeric insulin as a meansfor improving the bioavailability of inhaled insulin,” 30 Mar. 1999;U.S. Pat. No. 5,873,358A, entitled “Method of maintaining a diabeticpatient's blood glucose level in a desired range,” 23 Feb. 1999; U.S.Pat. No. 5,747,642A, entitled “Monomeric insulin analog formulations,” 5May 1998; U.S. Pat. No. 5,693,609A, entitled “Acylated insulin compoundanalogs,” 2 Dec. 1997; U.S. Pat. No. 5,650,486A, entitled “Monomericinsulin analog formulations,” 22 Jul. 1997; U.S. Pat. No. 5,646,242A,entitled “Selective acylation of epsilon-amino groups,” 8 Jul. 1997;U.S. Pat. No. 5,597,893A, entitled “Preparation of stable insulin analogcrystals,” 28 Jan. 1997; U.S. Pat. No. 5,547,929A, entitled “Insulinanalog formulations,” 20 Aug. 1996; U.S. Pat. No. 5,504,188A, entitled“Preparation of stable zinc insulin compound analog crystals,” 2 Apr.1996; U.S. Pat. No. 5,474,978A, entitled “Insulin analog formulations,”12 Dec. 1995; U.S. Pat. No. 5,461,031A, entitled “Monomeric insulinanalog formulations,” 24 Oct. 1995; U.S. Pat. No. 4,421,685A, entitled“Process for producing an insulin,” 20 Dec. 1983; U.S. Pat. No.6,221,837, entitled “Insulin derivatives with increased zinc binding” 24Apr. 2001; U.S. Pat. No. 5,177,058, entitled “Pharmaceutical formulationfor the treatment of diabetes mellitus” 5 Jan. 1993 (describespharmaceutical formulations including an insulin compound derivativemodified with a base at B31 and having an isoelectric point between 5.8and 8.5 and/or at least one of its physiologically tolerated salts in apharmaceutically acceptable excipient, and a relatively high zinc ioncontent in the range from above 1 μg to about 200 μg of zinc/IU,including insulin compound-B31-Arg-OH and humaninsulin-B31-Arg-B32-Arg-OH). The entire disclosure of each of theforegoing patent documents is incorporated herein by reference,particularly for teaching about the making, using and compositions ofvarious insulin compound analogs.

Insulin compound used to prepare the cation-insulin compound conjugatescan be prepared by any of a variety of recognized peptide synthesistechniques, e.g., classical (solution) methods, solid phase methods,semi-synthetic methods, and recombinant DNA methods. For example, Chanceet al., U.S. patent application Ser. No. 07/388,201, EPO383472, Brangeet al., EPO214826, and Belagaje et al., U.S. Pat. No. 5,304,473 disclosethe preparation of various proinsulin compound and insulin compoundanalogs and are herein incorporated by reference. The A and B chains ofthe insulin compound analogs may also be prepared via a proinsulincompound-like precursor molecule or single chain insulin compoundprecursor molecule using recombinant DNA techniques. See Frank at al.,“Peptides: Synthesis-Structure-Function,” Proc. Seventh Am. Pept. Symp.,Eds. D. Rich and E. Gross (1981); Bernd Gutte, Peptides: Synthesis,Structures, and Applications, Academic Press (Oct. 19, 1995); Chan, Wengand White, Peter (Eds.), Fmoc Solid Phase Peptide Synthesis: A PracticalApproach, Oxford University Press (March 2000); the entire disclosuresof which are incorporated herein by reference for their teachingsconcerning peptide synthesis, recombinant production and manufacture.

7.2 Modifying Moiety

The cation-insulin compound conjugate complexes include a modifyingmoiety coupled (e.g., covalently or ionically) to the insulin compoundto provide the insulin compound conjugate. Modifying moieties aremoieties coupled to the insulin compound that provide the insulincompound with desired properties as described herein. For example, themodifying moiety can reduce the rate of degradation of the insulincompound in various environments (such as the GI tract, and/or thebloodstream), such that less of the insulin compound is degraded in themodified form than would be degraded in the absence of the modifyingmoiety in such environments. Preferred modifying moieties are thosewhich permit the insulin compound conjugate to retain a therapeuticallysignificant percentage of the biological activity of the parent insulincompound. Further, preferred modifying moieties are those which areamphiphilic or hydrophilic, and/or which render the insulin compoundconjugate amphiphilic or hydrophilic or less lipophilic than ascientifically acceptable control, such as a corresponding insulincompound, or a corresponding unconjugated insulin compound.

Examples of suitable modifying moieties and insulin compound conjugatesuseful in the cation-insulin compound conjugate compositions can befound in the following patents, the entire disclosures of which areincorporated herein by reference: U.S. Pat. No. 6,303,569, entitled“Trialkyl-lock-facilitated polymeric prodrugs of amino-containingbioactive agents,” 16 Oct. 2001; U.S. Pat. No. 6,214,330, “Coumarin andrelated aromatic-based polymeric prodrugs,” 10 Apr. 2001; U.S. Pat. No.6,113,906, entitled “Water-soluble non-antigenic polymer linkable tobiologically active material,” 5 Sep. 2000; U.S. Pat. No. 5,985,263,entitled “Substantially pure histidine-linked protein polymerconjugates,” 16 Nov. 1999; U.S. Pat. No. 5,900,402, entitled “Method ofreducing side effects associated with administration of oxygen-carryingproteins,” 4 May 1999; U.S. Pat. No. 5,681,811, “Conjugation-stabilizedtherapeutic agent compositions, delivery and diagnostic formulationscomprising same, and method of making and using the same” 28 Oct. 1997;U.S. Pat. No. 5,637,749, entitled “Aryl imidate activated polyalkyleneoxides,” 10 Jun. 1997; U.S. Pat. No. 5,612,460, entitled “Activecarbonates of polyalkylene oxides for modification of polypeptides,” 18Mar. 1997; U.S. Pat. No. 5,567,422, entitled “Azlactone activatedpolyalkylene oxides conjugated to biologically active nucleophiles,” 22Oct. 1996; U.S. Pat. No. 5,405,877, entitled “Cyclic imide thioneactivated polyalkylene oxides,” 11 Apr. 1995; and U.S. Pat. No.5,359,030, entitled “Conjugation-stabilized polypeptide compositions,therapeutic delivery and diagnostic formulations comprising same, andmethod of making and using the same,” 25 Oct. 1994. Additional examplesof conjugated polypeptides useful in the formulations of the instantinvention can be found in the following U.S. patent applications, theentire specifications of which are incorporated herein by reference:U.S. patent application Ser. No. 09/134,803, filed 14 Aug. 1998; Ser.No. 10/018,879, filed 19 Dec. 2001; Ser. No. 10/235,381, filed 5 Sep.2002; Ser. No. 10/235,284, filed 5 Sep. 2002; and Ser. No. 09/873,797,filed 4 Jun. 2001. The entire disclosure of each of the foregoingpatents and patent applications is incorporated herein by reference fortheir teachings concerning moieties used to modify polypeptides.

The modifying moieties may include weak or degradable linkages in theirbackbones. For example, the PAGs can include hydrolytically unstablelinkages, such as lactide, glycolide, carbonate, ester, carbamate andthe like, which are susceptible to hydrolysis. This approach allows thepolymers to be cleaved into lower molecular weight fragments. Examplesof such polymers are described, for example, in U.S. Pat. No. 6,153,211,entitled, to Hubbell et al., the entire disclosure of which isincorporated herein by reference. See also U.S. Pat. No. 6,309,633, toEkwuribe et al., the entire disclosure of which is incorporated hereinby reference.

The modifying moiety can include any hydrophilic moieties, lipophilicmoieties, amphiphilic moieties, salt-forming moieties, and combinationsthereof. Representative hydrophilic, amphiphilic, and lipophilicpolymers and modifying moieties are described in more detail below.

7.2.1 Hydrophilic Moieties

Examples of suitable hydrophilic moieties include PAG moieties, otherhydrophilic polymers, sugar moieties, polysorbate moieties, andcombinations thereof.

7.2.2 Polyalkylene Glycol Moieties

PAGs are compounds with repeat alkylene glycol units. In someembodiments, the units are all identical (e.g., PEG or PPG). In otherembodiments, the alkylene units are different (e.g.,polyethylene-co-propylene glycol, or PLURONICS®). The polymers can berandom copolymers (for example, where ethylene oxide and propylene oxideare co-polymerized) or branched or graft copolymers.

PEG is a preferred PAG, and is useful in biological applications becauseit has highly desirable properties and is generally regarded as safe(GRAS) by the Food and Drug Administration. PEG generally has theformula H—(CH₂CH₂O)_(n)—H, where n can range from about 2 to about 4000or more, though the capping moieties may vary, e.g., mono-methoxy ordi-hydroxy. PEG typically is colorless, odorless, water-soluble orwater-miscible (depending on molecular weight), heat stable, chemicallyinert, hydrolytically stable, and generally nontoxic. PEG is alsobiocompatible, and typically does not produce an immune response in thebody. Preferred PEG moieties include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, or more PEG subunits.

The PEG may be monodispersed, substantially monodispersed, purelymonodispersed or substantially purely monodispersed (e.g., as previouslydescribed by the applicants in U.S. patent application Ser. No.09/873,731 and U.S. Pat. No. 09/873,797, both filed 4 Jun. 2001, theentire disclosures of which are incorporated herein by reference) orpolydispersed. One advantage of using the relatively low molecularweight, monodispersed polymers is that they form easily definedconjugate molecules, which can facilitate both reproducible synthesisand FDA approval.

The PEG can be linear with a hydroxyl group at each terminus (beforebeing conjugated to the remainder of the insulin compound). The PEG canalso be an alkoxy PEG, such as methoxy-PEG (or mPEG), where one terminusis a relatively inert alkoxy group (e.g., linear or branched OC₁₋₆),while the other terminus is a hydroxyl group (that is coupled to theinsulin compound).

The PEG can also be branched, which can in one embodiment be representedas R(-PEG-_(n)OH)_(m) in which R represents a central (typicallypolyhydric) core agent such as pentaerythritol, sugar, lysine orglycerol, n represents the number of PEG subunits and can vary for eacharm and is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50and m represents the number of arms, and ranges from 2 to the maximumnumber of attachment sitesz on the core agent. Each branch can be thesame or different and can be terminated, for example, with ethers and/oresters. The number of arms m can range from three to a hundred or more,and one or more of the terminal hydroxyl groups can be coupled to theremainder of the insulin compound, or otherwise subject to chemicalmodification.

Other branched PEGs include those represented by the formula(CH₃O-PEG-)_(p)R—Z, where p equals 2 or 3, R represents a central coresuch as lysine or glycerol, and Z represents a group such as carboxylthat is subject to ready chemical activation. Still another branchedform, the pendant PEG, has reactive groups, such as carboxyls, along thePEG backbone rather than, or in addition to, the end of the PEG chains.Forked PEG can be represented by the formula PEG(-LCHX₂)_(n), where L isa linking group and X is an activated terminal group.

7.2.3 Sugar Moieties

The modifying moieties described herein can include sugar moieties. Ingeneral, the sugar moiety is a carbohydrate product of at least onesaccharose group. Representative sugar moieties include, but are notlimited to, glycerol moieties, mono-, di-, tri-, and oligosaccharides,and polysaccharides such as starches, glycogen, cellulose andpolysaccharide gums. Specific monosaccharides include C₆ and above(preferably C₆ to C₈) sugars such as glucose, fructose, mannose,galactose, ribose, and sedoheptulose; di- and trisaccharides includemoieties having two or three monosaccharide units (preferably C₅ to C₈)such as sucrose, cellobiose, maltose, lactose, and raffinose.Conjugation using sugar moieties is described in U.S. Pat. Nos.5,681,811, 5,438,040, and 5,359,030, the entire disclosures of which areincorporated herein by reference.

7.2.4 Polysorbate Moieties

The modifying moieties may include one or more polysorbate moieties.Examples include sorbitan esters, and polysorbate derivatized withpolyoxyethylene. Conjugation using polysorbate moieties is described inU.S. Pat. Nos. 5,681,811, 5,438,040, and 5,359,030, the entiredisclosures of which are incorporated herein by reference.

7.2.5 Biocompatible Water-Soluble Polycationic Moieties

In some embodiments, biocompatible water-soluble polycationic polymerscan be used. Biocompatible water-soluble polycationic polymers include,for example, any modifying moiety having protonated heterocyclesattached as pendant groups. “Water soluble” in this context means thatthe entire modifying moiety is soluble in aqueous solutions, such asbuffered saline or buffered saline with small amounts of added organicsolvents as cosolvents, at a temperature between 20 and 37° C. In someembodiments, the modifying moiety itself is not sufficiently soluble inaqueous solutions per se but is brought into solution by grafting withwater-soluble polymers such as PEG chains. Examples include polyamineshaving amine groups on either the modifying moiety backbone or themodifying moiety side chains, such as poly-L-Lys and other positivelycharged polyamino acids of natural or synthetic amino acids or mixturesof amino acids, including poly(D-Lys), poly(ornithine), poly(Arg), andpoly(histidine), and nonpeptide polyamines such as poly(aminostyrene),poly(aminoacrylate), poly(N-methyl aminoacrylate),poly(N-ethylaminoacrylate), poly(N,N-dimethyl aminoacrylate),poly(N,N-diethylaminoacrylate), poly(aminomethacrylate), poly(N-methylamino-methacrylate), poly(N-ethyl aminomethacrylate), poly(N,N-dimethylaminomethacrylate), poly(N,N-diethyl aminomethacrylate),poly(ethyleneimine), polymers of quaternary amines, such aspoly(N,N,N-trimethylaminoacrylate chloride),poly(methyacrylamidopropyltrimethyl ammonium chloride), and natural orsynthetic polysaccharides such as chitosan.

7.2.6 Other Hydrophilic Moieties

The modifying moieties may also include other hydrophilic polymers.Examples include poly(oxyethylated polyols) such as poly(oxyethylatedglycerol), poly(oxyethylated sorbitol), and poly(oxyethylated glucose);poly(vinyl alcohol) (“PVA”); dextran; carbohydrate-based polymers andthe like. The polymers can be homopolymers or random or block copolymersand terpolymers based on the monomers of the above polymers, linearchain or branched.

Specific examples of suitable additional polymers include, but are notlimited to, poly(oxazoline), difunctional poly(acryloylmorpholine)(“PAcM”), and poly(vinylpyrrolidone) (“PVP”). PVP and poly(oxazoline)are well known polymers in the art and their preparation will be readilyapparent to the skilled artisan. PAcM and its synthesis and use aredescribed in U.S. Pat. No. 5,629,384 and U.S. Pat. No. 5,631,322, thedisclosures of which are incorporated herein by reference in theirentirety.

7.2.7 Bioadhesive Polyanionic Moieties

Certain hydrophilic polymers appear to have potentially usefulbioadhesive properties. Examples of such polymers are found, forexample, in U.S. Pat. No. 6,197,346, to Mathiowitz, et al. Thosepolymers containing carboxylic groups (e.g., poly(acrylic acid)) exhibitbioadhesive properties, and are also readily conjugated with the insulincompounds described herein. Rapidly bioerodible polymers that exposecarboxylic acid groups on degradation, such aspoly(lactide-co-glycolide), polyanhydrides, and polyorthoesters, arealso bioadhesive polymers. These polymers can be used to deliver theinsulin compounds to the gastrointestinal tract. As the polymersdegrade, they can expose carboxylic acid groups to enable them to adherestrongly to the gastrointestinal tract, and can aid in the delivery ofthe insulin compound conjugates.

7.2.8 Lipophilic Moieties

In some embodiments, the modifying moieties include one or morelipophilic moieties. The lipophilic moiety may be various lipophilicmoieties as will be understood by those skilled in the art including,but not limited to, alkyl moieties, alkenyl moieties, alkynyl moieties,aryl moieties, arylalkyl moieties, alkylaryl moieties, fatty acidmoieties, adamantantyl, and cholesteryl, as well as lipophilic polymersand/or oligomers.

The alkyl moiety can be a saturated or unsaturated, linear, branched, orcyclic hydrocarbon chain. In some embodiments, the alkyl moiety has 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more carbon atoms.Examples include saturated, linear alkyl moieties such as methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl,nonadecyl and eicosyl; saturated, branched alkyl moieties such asisopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl,2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; andunsaturated alkyl moieties derived from the above saturated alkylmoieties including, but not limited to, vinyl, allyl, 1-butenyl,2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. In other embodiments,the alkyl moiety is a lower alkyl moiety. In still other embodiments,the alkyl moiety is a C₁ to C₃ lower alkyl moiety. In some embodiments,the modifying moiety specifically does not consist of an alkyl moiety,or specifically does not consist of a lower alkyl moiety, orspecifically does not consist of an alkane moiety, or specifically doesnot consist of a lower alkane moiety.

The alkyl groups can either be unsubstituted or substituted with one ormore substituents, and such substituents preferably either do notinterfere with the methods of synthesis of the conjugates or eliminatethe biological activity of the conjugates. Potentially interferingfunctionality can be suitably blocked with a protecting group so as torender the functionality non-interfering. Each substituent may beoptionally substituted with additional non-interfering substituents. Theterm “non-interfering” characterizes the substituents as not eliminatingthe feasibility of any reactions to be performed in accordance with theprocess of this invention.

The lipophilic moiety may be a fatty acid moiety, such as a natural orsynthetic, saturated or unsaturated, linear or branched fatty acidmoiety. In some embodiments, the fatty acid moiety has 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, ormore carbon atoms. In some embodiments, the modifying moietyspecifically does not consist of a fatty acid moiety; or specificallydoes not consist of a fatty acid moiety having 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or morecarbon atoms.

When the modifying moiety includes an aryl ring, the ring can befunctionalized with a nucleophilic functional group (such as OH, or SH)that is positioned so that it can react in an intramolecular fashionwith the carbamate moiety and assist in its hydrolysis. In someembodiments, the nucleophilic group is protected with a protecting groupcapable of being hydrolyzed or otherwise degraded in vivo, with theresult being that when the protecting group is deprotected, hydrolysisof the conjugate, and resultant release of the parent insulin compound,is facilitated.

Other examples of suitable modifying moieties include —C(CH₂OH)₃;—CH(CH₂OH)₂; —C(CH₃)₃; —CH(CH₃)₂.

7.2.9 Amphiphilic Moieties

In some embodiments, the modifying moiety includes an amphiphilicmoiety. Many polymers and oligomers are amphiphilic. These are oftenblock co-polymers, branched copolymers or graft co-polymers that includehydrophilic and lipophilic moieties, which can be in the form ofoligomers and/or polymers, such as linear chain, branched, or graftpolymers or co-polymers.

The amphiphilic modifying moieties may include combinations of any ofthe lipophilic and hydrophilic moieties described herein. Such modifyingmoieties typically include at least one reactive functional group, forexample, halo, hydroxyl, amine, thiol, sulfonic acid, carboxylic acid,isocyanate, epoxy, ester, and the like, which is often at a terminal endof the modifying moiety. These reactive functional groups can be used toattach a lipophilic linear or branched chain alkyl, alkenyl, alkynyl,arylalkyl, or alkylaryl group, or a lipophilic polymer or oligomer,thereby increasing the lipophilicity of the modifying moiety (andthereby rendering them generally amphiphilic).

The lipophilic groups can, for example, be derived from mono- ordi-carboxylic acids, or where appropriate, reactive equivalents ofcarboxylic acids such as anhydrides or acid chlorides. Examples ofsuitable precursors for the lipophilic groups are acetic acid, propionicacid, butyric acid, valeric acid, isobutyric acid, trimethylacetic acid,caproic acid, caprylic acid, heptanoic acid, capric acid, pelargonicacid, lauric acid, myristic acid, palmitic acid, stearic acid, behenicacid, lignoceric acid, ceratic acid, montanoic acid, isostearic acid,isononanoic acid, 2-ethylhexanoic acid, oleic acid, ricinoleic acid,linoleic acid, linolenic acid, erucic acid, soybean fatty acid, linseedfatty acid, dehydrated castor fatty acid, tall oil fatty acid, tung oilfatty acid, sunflower fatty acid, safflower fatty acid, acrylic acid,methacrylic acid, maleic anhydride, orthophthalic anhydride,terephthalic acid, isophthalic acid, adipic acid, azelaic acid, sebacicacid, tetrahydrophthalic anhydride, hexahydrophthalic anhydride,succinic acid and polyolefin carboxylic acids.

The terminal lipophilic groups need not be equivalent, i.e., theresulting copolymers can include terminal lipophilic groups that are thesame or different. The lipophilic groups can be derived from more thanone mono or di-functional alkyl, alkenyl, alkynyl, cycloalkyl, arylalkylor alkylaryl group as defined above.

7.2.10 PAG-Alkyl Modifying Moieties

The modifying moiety may be a linear or branched polymeric moiety havingone or more linear or branched PAG moieties and/or one or more linear orbranched, substituted or unsubstituted alkyl moieties. In certain cases,such moieties are considered amphiphilic; however, the PAG and alkylmoieties may be varied to render such moieties more lipophilic or morehydrophilic. In certain embodiments, the modifying moiety specificallydoes not consist of an alkyl moiety and in other embodiments, themodifying moiety specifically does not consist of an alkane moiety.

The PAG moieties in some embodiments include 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 PAGsubunits arranged in linear or branched form. The PAG moieties in someembodiments include PEG, PPG and/or PBG subunits. The alkyl moieties insome embodiments preferably have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkyl moieties arepreferably alkane moieties. The modifying moiety may include a cappingmoiety, such as —OCH₃. Further, the modifying moiety may include ahydrophobic group, such as a pivaloyl group.

In one embodiment, the modifying moiety has a formula:

where o, p and q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, or 50, and at least one of o, p and q is at least 2. X, Y and Zare independently selected from —C—, —O—, —C(O)—, —C(O)O—, —OC(O)—,—NH—, —NHC(O)—, and —C(O)NH—, and R is H or an alkyl, preferably a loweralkyl, more preferably methyl. The variables o, p and q are preferablyselected to yield a hydrophilic or amphiphilic modifying moiety, and arepreferably selected in relation to the insulin compound to yield ahydrophilic or amphiphilic insulin compound conjugate, preferably amonoconjugate, diconjugate or triconjugate. In one preferred embodimentfor an insulin compound conjugate which is to be used for basal insulincompound maintenance, o, p and q are selected to yield a PAG which isproximal to the insulin compound and an alkyl moiety which is distal tothe insulin compound. Alternatively, O, P and Q may be selected to yielda PAG which is distal to the insulin compound and an alkyl which isproximal to the insulin. In an alternative embodiment, R is a pivaloylgroup or an alkyl-pivaloyl group.

In a related embodiment, the modifying moiety has a formula:

where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25 and n is from 2 to 100, preferably 2to 50, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, X is —C—, —O—, —C(O)—, —NH—,—NHC(O)—, or —C(O)NH—, and Y is lower alkyl or —H. X is preferably O andY is preferably —CH₃. In some cases the carbonyl group (—C(O)—) may beabsent, and the —(CH2)- moiety may be coupled to an available group onan amino acid, such as a hydroxyl group or a free carboxylic acid group.

In a preferred embodiment, the modifying moiety has a structure selectedfrom the following:

and

(when the immediately preceding modifying moiety is coupled to humaninsulin at B29, the resulting monoconjugate is referred to as IN105).

(when the immediately preceding modifying moiety is coupled to humaninsulin at B29, the resulting monoconjugate is referred to as HIM2). Anyof the foregoing moieties may, for example, be coupled to human insulinat a nucleophilic residue, e.g., A1, B1 or B29. In some cases thecarbonyl group (—C(O)—) may be absent or replaced with an alkyl moiety,preferably a lower alkyl moiety, and the —(CH₂)— moiety may be coupledto an available group on an amino acid, such as a hydroxyl group or afree carboxylic acid group.

In another embodiment, the modifying moiety has a formula:

where each C is independently selected and is an alkyl moiety having mcarbons and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20; and each PAG is independently selected and is a PAGmoiety having n subunits and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; each X isindependently selected and is a linking moiety coupling PAG to C, and ispreferably —C—, —O—, —C(O)—, —NH—, —NHC(O)—, or —C(O)NH—. In someembodiments the C_(m)—X moiety is absent, and the PAG_(n) moiety isterminated with an —OH moiety or an —OCH₃ moiety. For example, the PAGmay be methoxy-terminated or hydroxy-terminated PAG, having 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 PAGsubunits, including PEG, PPG, and/or PBG subunits. In some cases thecarbonyl group (—C(O)—) may be replaced with an alkyl moiety, preferablya lower alkyl moiety, which may be coupled to an available group on anamino acid, such as a hydroxyl group or a free carboxylic acid group.

The modifying moiety may, for example, have a formula:

where each C is independently selected and is an alkyl moiety having mcarbons and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20; and each PAG is independently selected and is a PAGmoiety having n subunits and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; X is —O—, or —NH—;each o is independently selected and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or 15. For example, the PAG may have 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 PAG subunits,including PEG, PPG, and/or PBG subunits. In some cases the carbonylgroup (—C(O)—) proximal to the point of attachment may be absent orreplaced with an alkyl moiety, preferably a lower alkyl moiety, and the—(CH2)- moiety may be coupled to an available group on an amino acid,such as a hydroxyl group or a free carboxylic acid group.

The modifying moiety may, for example, have a formula:

where each C is independently selected and is an alkyl moiety having mcarbons and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20; and each PAG is independently selected and is a PAGmoiety having n subunits and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; each X isindependently selected and is a linking moiety coupling PAG to C, and ispreferably —C—, —O—, —C(O)—, —NH—, —NHC(O)—, or —C(O)NH—; each o isindependently selected and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15. In some embodiments the Cm-X moiety is absent, and the PAGmoiety is terminated with an —OH moiety or an —OCH₃ moiety. For example,the PAG may be methoxy-terminated or hydroxy-terminated PAG, having 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 PAGsubunits, including PEG, PPG, and/or PBG subunits. In some cases thecarbonyl group (—C(O)—) proximal to the point of attachment may beabsent, and the —(CH2)- moiety may be coupled to an available group onan amino acid, such as a hydroxyl group or a free carboxylic acid group.

In another embodiment, the modifying moiety may have a formula:

—X—R¹—Y-PAG-Z—R²   (Formula VI)

where,

X, Y and Z are independently selected linking groups and each isoptionally present, and X, when present, is coupled to the insulincompound by a covalent bond,

at least one of R¹ and R² is present, and is lower alkyl and mayoptionally include a carbonyl group,

R² is a capping group, such as —CH₃, —H, tosylate, or an activatinggroup, and

PAG is a linear or branched carbon chain incorporating one or morealkalene glycol moieties (i.e., oxyalkalene moieties), and optionallyincorporating one or more additional moieties selected from the groupconsisting of —S—, —O—, —N—, and —C(O)—, and

where the modifying moiety has a maximum number of 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 heavyatoms.

In embodiments of the invention, any one or more of X, Y and Z may beabsent. Further, when present, X, Y and/or Z may be independentlyselected from —C(O)—, —O—, —S—, —C— and —N—. In one embodiment, Z is—C(O)—. In another embodiment, Z is not present.

In some embodiments, R¹ is lower alkyl, and R² is not present. In otherembodiments, R² is lower alkyl, and R¹ is not present.

In another embodiment, the modifying moiety may include a linear orbranched, substituted carbon chain moiety having a backbone of 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24or 25 atoms selected from the group consisting of —C, —C—, —O—, ═O, —S—,—N—, —Si—. The heavy atoms will typically include one or more carbonatoms and one or more non-carbon heavy atoms selected from the groupconsisting of —O—, —S—, —N—, and ═O. The carbon atoms and non-carbonheavy atoms are typically present in a ratio of at least 1 carbon atomfor every non-carbon heavy atom, preferably at least 2 carbon atoms forevery non-carbon heavy atom, more preferably at least 3 carbon atoms forevery non-carbon heavy atom. The carbon atoms and oxygen atoms aretypically present in a ratio of at least 1 carbon atom for every oxygenatom, preferably at least 2 carbon atoms for every oxygen atom, morepreferably at least 3 carbon atoms for every oxygen atom. The modifyingmoiety may include one or more capping groups, such as branched orlinear C₁₋₆, branched or linear, or a carbonyl. The modifying moietywill typically include hydrogens, and one or more of the hydrogens maybe substituted with a fluorine (which is a heavy atom but should not becounted as a heavy atom in the foregoing formula). The modifying moietymay in some cases specifically exclude unsubstituted alkyl moieties. Themodifying moiety may, for example, be coupled to an available group onan amino acid, such as an amino group, a hydroxyl group or a freecarboxylic acid group the polypeptide, e.g., by a linking group, such asa carbamate, carbonate, ether, ester, amide, or secondary amine group,or by a disulfide bond. The molecules in the linking group are countedas part of the modifying moiety. In a preferred embodiment, themolecular weight of the modifying moiety is less than the molecularweight of the HIM2 modifying moiety.

The invention includes modifying moieties having a formula:

where n is 1, 2, 3 or 4, and m is 1, 2, 3, 4 or 5.

The invention includes modifying moieties having a formula:

where n is 1, 2, 3, 4 or 5, and m is 1, 2, 3 or 4.

The invention includes modifying moieties having a formula:

where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20.

The invention also includes modifying moieties having a formula:

where PAG is a PAG moiety having m subunits and m is 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and n is 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

Other preferred modifying moieties include:

The following modifying moieties can be particularly preferred for usein a basal insulin compound replacement regimen.

Still other modifying moieties include the following:

R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃, R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₃, R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—O—CH₂—CH₃, 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R—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃, R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—O—CH₃, R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₂—CH₃, R—CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH₃,R—CH₂—O—CH₂—CH₂—O—CH₃, R—CH₂—O—CH₂—CH₃, R—CH₂—O—CH₃,

where R is —H, —OH, —CH₂OH, —CH(OH)₂, —C(O)OH, —CH₂C(O)OH, or anactivating moiety, such as a carbodiimide, a mixed anhydride, or anN-hydroxysuccinimide, or a capping group. The invention also includessuch moieties attached to a protein or peptide, preferably to an insulincompound. Specific conjugation strategies are discussed in more detailbelow. Of these modifying moieties, preferred moieties are those whichrender the insulin compound less lipophilic and/or more hydrophilic thanthe corresponding unconjugated insulin compound. The invention includessuch modifying moieties further including one or more carbonyl groups,preferably 1, 2, 3, 4, or 5 carbonyl groups; the carbonyl groups may beinserted into the modifying moiety, or an —O— or —CH₂— may be replacedwith a carbonyl. Further, any of the —CH₂— or —CH₃ moieties may besubstituted, e.g., with a lower alkyl or an —OH or a PAG chain having 1,2, 3, 4, or 5 PAG subunits, which may be the same or different.Preferably R is selected so that each —O— is separated from the nearest—O— by at least 2 carbons. The invention also includes branchedmodifying moieties in which two or more of the moieties are attached toa branching moiety, such as a lysine.

The pharmaceutical characteristics, such as hydrophilicity/lipophilicityof the conjugates according to embodiments of the invention, can bevaried by, for example, adjusting the lipophilic and hydrophilicportions of the modifying moieties, e.g., by increasing or decreasingthe number of PAG monomers, the type and length of alkyl chain, thenature of the PAG-peptide linkage, and the number of conjugation sites.The exact nature of the modifying moiety-peptide linkage can be variedsuch that it is stable and/or sensitive to hydrolysis at physiologicalpH or in plasma. The invention also includes any of the foregoingmodifying moieties coupled to a polypeptide, preferably to insulincompound. Preferably, the modifying moiety renders the polypeptide moresoluble than a corresponding unconjugated polypeptide, e.g., by amultiplier of at least 1.05, 1.25. 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13,13.5, 14, 14.5, or 15. A modifying moiety of the invention may becoupled, for example, to an insulin compound, such as a human insulin,at any available point of attachment. A preferred point of attachment isa nucleophilic residue, e.g., A1, B1 and/or B29.

Moreover, it will be appreciated that one aspect of the inventionincludes novel modifying moieties, such as but not limited to themoieties of Formulas VII and VIII, in a carboxylic acid form. Further,where the modifying moiety includes a carboxyl group, it can beconverted to a mixed anhydride and reacted with an amino group of apeptide to create a conjugate containing an amide bond. In anotherprocedure, the carboxyl group can be treated with water-solublecarbodiimide and reacted with the peptide to produce conjugatescontaining amide bonds. Consequently, the invention includes activatedforms of the novel moieties presented herein, such as activated forms ofthe modifying moieties of Formulas VII and VIII and other noveloligomers of the invention, such as carbodiimides, mixed anhydrides, orN-hydroxysuccinimides.

In some cases, the modifying moiety may be coupled to the polypeptidevia an amino acid or series of 2 or more amino acids coupled to theC-terminus, or a side chain of the polypeptide. For example, in oneembodiment, the modifying moiety is coupled at the —OH or —C(O)OH ofThr, and the mm-modified Thr is coupled to a polypeptide at the carboxyterminus. For example, in one embodiment, the modifying moiety iscoupled at the —OH or —C(O)OH of Thr, and the modified Thr is coupled tothe B29 amino acid (e.g., a B29 Lys for human insulin) of des-Thrinsulin compound. In another example, the mm is coupled at the —OH or—C(O)OH of Thr of a terminal octapeptide from the insulin compoundB-chain, and the mm-modified octapeptide is coupled to the B22 aminoacid of des-octa insulin compound. Other variations will be apparent toone skilled in the art in light of this specification.

7.2.11 Salt-Forming Moieties

In some embodiments, the modifying moiety comprises a salt-formingmoiety. The salt-forming moiety may be various suitable salt-formingmoieties as will be understood by those skilled in the art including,but not limited to, carboxylate and ammonium. In some embodiments wherethe modifying moiety includes a salt forming moiety, the insulincompound conjugate is provided n salt form. In these embodiments, theinsulin compound conjugate is associated with a suitablepharmaceutically acceptable counterion as will be understood by thoseskilled in the art including, but not limited to, negative ions such aschloro, bromo, iodo, phosphate, acetate, carbonate, sulfate, tosylate,and mesylate, or positive ions such as sodium, potassium, calcium,lithium, and ammonium.

The foregoing examples of modifying moieties are intended asillustrative and should not be taken as limiting in any way. One skilledin the art will recognize that suitable moieties for conjugation toachieve particular functionality will be possible within the bounds ofthe chemical conjugation mechanisms disclosed and claimed herein.Accordingly, additional moieties can be selected and used according tothe principles as disclosed herein.

7.3 Conjugation Strategies

Factors such as the degree of conjugation with modifying moieties,selection of conjugation sites on the molecule and selection ofmodifying moieties may be varied to produce a conjugate which, forexample, is less susceptible to in vivo degradation, and thus, has anincreased plasma half life. For example, the insulin compounds may bemodified to include a modifying moiety at one, two, three, four, five,or more sites on the insulin compound structure at appropriateattachment (i.e., modifying moiety conjugation) sites suitable forfacilitating the association of a modifying moiety thereon. By way ofexample, such suitable conjugation sites may comprise an amino acidresidue, such as a lysine amino acid residue.

In some embodiments, the insulin compound conjugates are monoconjugates.In other embodiments, the insulin compound conjugates aremulti-conjugates, such as di-conjugates, tri-conjugates,tetra-conjugates, penta-conjugates and the like. The number of modifyingmoieties on the insulin compound is limited only by the number ofconjugation sites on the insulin compound. In still other embodiments,the insulin compound conjugates are a mixture of mono-conjugates,di-conjugates, tri-conjugates, tetra-conjugates, and/orpenta-conjugates. Preferred conjugation strategies are those which yielda conjugate relating some or all of the bioactivity of the parentinsulin compound.

Preferred attachment sites include A1 N-terminus, B1 N-terminus, and B29lysine side chain. The B29 monoconjugate and B1, B29 diconjugates arehighly preferred. Another preferred point of attachment is an aminofunctionality on a C-peptide component or a leader peptide component ofthe insulin compound.

One or more modifying moieties (i.e., a single or a plurality ofmodifying moiety structures) may be coupled to the insulin compound. Themodifying moieties in the plurality are preferably the same. However, itis to be understood that the modifying moieties in the plurality may bedifferent from one another, or, alternatively, some of the modifyingmoieties in the plurality may be the same and some may be different.When a plurality of modifying moieties are coupled to the insulincompound, it may be preferable to couple one or more of the modifyingmoieties to the insulin compound with hydrolyzable bonds and couple oneor more of the modifying moieties to the insulin compound withnon-hydrolyzable bonds. Alternatively, all of the bonds coupling theplurality of modifying moieties to the insulin compound may behydrolyzable but have varying degrees of hydrolyzability such that, forexample, one or more of the modifying moieties may be relatively rapidlyremoved from the insulin compound by hydrolysis in the body and one ormore of the modifying moieties is more slowly removed from the insulincompound by hydrolysis in the body.

7.3.1 Coupling of Modifying Moiety to Insulin Compound

The modifying moiety is preferably covalently coupled to the insulincompound. More than one moiety on the modifying moiety may be covalentlycoupled to the insulin compound. Coupling may employ hydrolyzable ornon-hydrolyzable bonds or mixtures of the two (i.e., different bonds atdifferent conjugation sites).

In some embodiments, the insulin compound is coupled to the modifyingmoiety using a hydrolyzable bond (e.g., an ester, carbonate orhydrolyzable carbamate bond). Use of a hydrolyzable coupling willprovide an insulin compound conjugate that acts as a prodrug. A prodrugapproach may be desirable where the insulin compound-modifying moietyconjugate is inactive (i.e., the conjugate lacks the ability to affectthe body through the insulin compound's primary mechanism of action),such as when the modifying moiety conjugation site is in a bindingregion of insulin compound. Use of a hydrolyzable coupling can alsoprovide for a time-release or controlled-release effect, administeringthe insulin compound over a given time period as one or more modifyingmoieties are cleaved from their respective insulin compound-modifyingmoiety conjugates to provide the active drug.

In other embodiments, the insulin compound is coupled to the modifyingmoiety utilizing a non-hydrolyzable bond (e.g., a non-hydrolyzablecarbamate, amide, or ether bond). Use of a non-hydrolyzable bond may bepreferable when it is desirable to allow therapeutically significantamounts of the insulin compound conjugate to circulate in thebloodstream for an extended period of time, e.g., at least 2 hours postadministration. Bonds used to covalently couple the insulin compound tothe modifying moiety in a non-hydrolyzable fashion are typicallyselected from the group consisting of covalent bond(s), ester moieties,carbonate moieties, carbamate moieties, amide moieties and secondaryamine moieties.

The modifying moiety may be coupled to the insulin compound at variousnucleophilic residues, including, but not limited to, nucleophilichydroxyl functions and/or amino functions. Nucleophilic hydroxylfunctions may be found, for example, at serine and/or tyrosine residues,and nucleophilic amino functions may be found, for example, at histidineand/or Lys residues, and/or at the one or more N-terminus of the A or Bchains of the insulin compound. When a modifying moiety is coupled tothe N-terminus of the natriuretic peptide, coupling preferably forms asecondary amine

The modifying moiety may be coupled to the insulin compound at a free—SH group, e.g., by forming a thioester, thioether or sulfonate bond.

The modifying moiety may be coupled to the insulin compound via one ormore amino groups. Examples in human insulin include the amino groups atA1, B1 and B29. In one embodiment, a single modifying moiety is coupledto a single amino group on the insulin compound. In another embodiment,two modifying moieties are each connected to a different amino group onthe insulin compound. Where there are two modifying moieties coupled totwo amino groups, a preferred arrangement is coupling of at B1 and B29.Where there are multiple polymers, the polymers may all be the same oror one or more of the polymers may be different from the others. Variousmethods and types of coupling of polymers to insulin compounds aredescribed in U.S. patent application Ser. No. 09/873,899, entitled“Mixtures of insulin compound conjugates comprising polyalkylene glycol,uses thereof, and methods of making same,” filed on 4 Jun. 2001, theentire disclosure of which is incorporated herein by reference.

In still other embodiments, a partial prodrug approach may be used, inwhich a portion of the modifying moiety is hydrolyzed. For example, seeU.S. Pat. No. 6,309,633 to Ekwuribe et al. (the entire disclosure ofwhich is incorporated herein by reference), which describes modifyingmoieties having hydrophilic and lipophilic components in which thelipophilic components hydrolyze in vivo to yield a micropegylatedconjugate.

7.3.2 Selection of Modifying Moiety and Properties of theInsulin-Compound Conjugate and Complexes Thereof

The modifying moiety may be selected to provide desired attributes tothe insulin compound conjugate and complexes thereof. Preferredmodifying moieties are selected to render the insulin compound moresoluble in an aqueous solution than the aqueous solubility of theinsulin compound in the absence of the modifying moiety, preferably atleast 1.05, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,or 15 times more soluble than the parent insulin compound (i.e., thecorresponding unconjugated insulin compound) in an aqueous solution. Forexample, uncomplexed native human insulin has a solubility of ˜18 mg/mlat a pH of about 7.4. The inventors have surprisingly discovered amethod of complexing human insulin conjugates that are more soluble thanhuman insulin by a multiplier of at least 1.05, 1.25, 1.5, 1.75, 2, 2.5,3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15.

In certain embodiments, the modifying moiety is selected to render aninsulin compound conjugate having an aqueous solubility that exceeds 1g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 20 g/L, 50 g/L, 100 g/L, or even 150g/L at a pH ranging from about 4 to about 8, preferably preferably a pHranging from about 5 to about 7.5, ideally pH of about 7.4.

The insulin compound conjugate can be more orally bioavalable in amammal than a scientifically acceptable control, such as a correspondingunconjugated insulin compound. In other embodiments, the insulincompound conjugate is more orally bioavalable in a human than ascientifically acceptable control, such as a corresponding unconjugatedinsulin compound. In certain embodiments, absorption of the insulincompound conjugate, e.g., as measured by plasma levels of the conjugate,is at least 1.5, 2, 2.5, 3, 3.5, or 4 times greater that the absorptionof an unconjugated insulin compound control.

It will be appreciated that while in some aspects of the invention themodifying moiety is selected to render the insulin compound conjugatemore soluble than a corresponding unconjugated insulin compound, inother aspects the modifying moiety may also or alternatively be selectedto render the insulin compound conjugate equally or more hydrophilicthan a corresponding unconjugated insulin compound. Further, themodifying moiety may be selected to render the insulin compoundconjugate more amphiphilic than a corresponding unconjugated insulincompound.

In some embodiments, the cation-insulin compound conjugate complex isequally as water soluble or more water soluble than (a) a correspondinguncomplexed insulin compound conjugate, (b) a corresponding uncomplexedand unconjugated insulin compound, and/or (c) a corresponding complexedbut unconjugated insulin compound.

In a preferred embodiment, the water solubility of the insulin compoundconjugate is decreased by the addition of Zn⁺⁺. In some embodiments, themodifying moiety is selected to render the insulin compound conjugateequally or more soluble than a corresponding unconjugated insulincompound, and the water solubility of the insulin compound conjugate isdecreased by the addition of zinc. In other embodiments, the modifyingmoiety is selected to render the insulin compound conjugate equally ormore soluble than a corresponding unconjugated insulin compound, thewater solubility of the insulin compound conjugate is decreased by theaddition of zinc, and the water solubility of the cation complex isgreater than the water solubility of insulin compound. In anotheraspect, the insulin compound conjugate is a fatty acid acylated insulincompound, the cation is zinc, and the water solubility of the insulincompound conjugate is decreased by the addition of the zinc. In stillanother embodiment, the insulin compound conjugate is a fatty acidacylated insulin compound that is equally or more water soluble than acorresponding unconjugated insulin compound, the cation is zinc, and thewater solubility of the insulin compound conjugate is decreased by theaddition of the zinc.

In certain preferred embodiments, the lipophilicity of the insulincompound conjugate relative to the corresponding parent insulin compoundis 1 or less than 1. The relative lipophilicity of the insulin compoundconjugate as compared to corresponding parent insulin compound (k_(rel))can, for example, be determined as follows:krel=(t_(conjugate)−t₀)/(t_(human)−t₀), where relative lipophilicity ismeasured on an LiChroSorb RP18 (5 μm, 250×4 mm) high performance liquidchromatography column by isocratic elution at 40° C. The followingmixtures can be used as eluents: 0.1M sodium phosphate buffer at pH 7.3containing 10% acetonitrile, and 50% acetonitrile in water. Void time(t₀) is identified by injecting 0.1 mM sodium nitrate. Retention timefor human insulin is adjusted to at least 2t₀ by varying the rationbetween the mixtures of (c)(i) and (c)(ii). Preferably, in theseembodiments, the relative lipophilicity is about equal to 1 or is lessthan 1 or substantially less than 1. In a preferred embodiment, theinsulin compound is human insulin, and the relative lipophilicity isless than 1. Preferably the relative lipophilicity is less than about0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, or 0.90.Discussion of techniques for determining solubility and/or lipophilicityof insulin and insulin conjugates are set forth in the U.S. Pat. No.5,750,499 entitled “Acylated insulin” issued to Harelund et al., on 12May 1998, the entire disclosure of which is incorporated herein byreference.

In one embodiment, the relative lipophilicity is as described above andthe modifying moiety is a carbon chain having 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17 or 18 carbons, wherein the carbon chain comprises 2,3, 4, 5, 6, 7, 8, 9, or 10 oxy groups inserted therein. In anotherembodiment, the relative lipophilicity is as described above and themodifying moiety is a carbon chain having 5, 6, 7, 8, 9 or 10 carbons,wherein the carbon chain comprises 2, 3 or 4 oxy groups insertedtherein. In a related embodiment, the relative lipophilicity is asdescribed above and the modifying moiety comprises 2, 3, 4, 5, 6, 7, 8,9 or 10 polyalkalene glycol units. In another related embodiment,relative lipophilicity is as described above and the modifying moietycomprises 1, 2 or 3 polyethylene glycol units and 1, 2 or 3polypropylene glycol units.

7.4 Metal Cation Component and Characteristics of Complexes

The cation-insulin compound conjugate complexes include a metal cation.Suitable metal cations for use as the cation component include any metalcation capable of complexing, aggregating, or crystallizing with theinsulin compound conjugate. It is preferred that the metal cation becomplexed to the insulin compound conjugate. Single or multiple cationscan be used. The cation is preferably not significantly oxidizing to theinsulin compound conjugate, i.e., not oxidizing to the extent that thecomplexes are rendered useless for their intended purpose.

In some embodiments, the metal cation is biocompatible. A metal cationis biocompatible if the cation presents no unduly significantdeleterious effects on the recipient's body, such as a significantimmunological reaction at the injection site. However, it will beappreciated that in some circumstances, the risks of toxity and otherdeleterious effects may be outweighed by the benefits of thecation-insulin compound conjugate composition, and therefore may beacceptable under such circumstances.

The suitability of metal cations for stabilizing biologically activeagents and the ratio of metal cation to biologically active agent neededcan be determined by one of ordinary skill in the art by performing avariety of tests for stability such as polyacrylamide gelelectrophoresis, isoelectric focusing, reverse phase chromatography, andHPLC analysis on particles of metal cation-stabilized biologicallyactive agents prior to and following particle size reduction and/orencapsulation.

The metal cation component suitably includes one or more monovalent,divalent, or trivalent metal cations, or combinations thereof. In apreferred embodiment, the metal cation is a Group II or transition metalcation. Examples of suitable divalent cations include Zn⁺⁺, Mn⁺⁺, Ca⁺⁺,Fe⁺⁺, Ni⁺⁺, Cu⁺, Co⁺⁺ and/or Mg⁺. Where a monovalent cation is included,it is preferably Na⁺, Li⁺, or K⁺. The cation is preferably added as asalt, such as a chloride or acetate salt, most preferred are ZnCl₂ andZnAc.

The molar ratio of insulin compound conjugate to cation is typicallybetween about 1:1 and about 1:100, preferably between about 1:2 andabout 1:12, and more preferably between about 1:2 and about 1:7 or about1:2, 1:3, 1:4, 1:5, 1:6, or 1:7. In a particular embodiment, Zn⁺⁺ isused as the cation component, it is provided at a zinc cation componentto insulin compound conjugate molar ratio of about 1:1 and about 1:100,preferably between about 1:2 and about 1:12, and more preferably betweenabout 1:2 and about 1:7 or about 1:2, 1:3, 1:4, 1:5, 1:6, or 1:7.

The cation component is preferably greater than about 90% a singlecation, such as Zn⁺⁺. Preferably, the cation is greater than about 95%,99%, or 99.9% Zn⁺⁺.

Preferably resistance of the complexed insulin compound conjugate tochymotrypsin degradation is greater than the chymotrypsin degradation ofthe corresponding uncomplexed insulin compound conjugate. Preferablyresistance of the complexed insulin compound conjugate to chymotrypsindegradation is greater than the chymotrypsin degradation of thecorresponding complexed but unconjugated insulin compound.

The complexed insulin compound conjugate can be more orally bioavalablein a mammal than a scientifically acceptable control, such as acorresponding uncomplexed insulin compound conjugate. In otherembodiments, the complexed insulin compound conjugate is more orallybioavalable in a human than a scientifically acceptable control, such asa corresponding uncomplexed insulin compound conjugate. In certainembodiments, absorption of the complexed insulin compound conjugate,e.g., as measured by plasma levels of the conjugate, is at least 1.5, 2,2.5, 3, 3.5, or 4 times greater that the absorption of an uncomplexedinsulin compound conjugate.

The complexed insulin compound conjugate can be more orally bioavalablein a mammal than a scientifically acceptable control, such as acorresponding complexed but unconjugated insulin compound. In otherembodiments, the complexed insulin compound conjugate is more orallybioavalable in a human than a scientifically acceptable control, such asa corresponding complexed but unconjugated insulin compound. In certainembodiments, absorption of the complexed insulin compound conjugate,e.g., as measured by plasma levels of the conjugate, is at least 1.5, 2,2.5, 3, 3.5, or 4 times greater that the absorption of an complexed butunconjugated insulin compound.

7.5 Complexing Agents

In some embodiments, the cation-insulin compound conjugate compositionsinclude one or more complexing agents. Examples of complexing agentssuitable for use in the present invention include protamines, surfen,globin proteins, spermine, spermidine albumin, amino acids, carboxylicacids, polycationic polymer compounds, cationic polypeptides, anionicpolypeptides, nucleotides, and antisense. See Brange, J., Galenics ofInsulin compound, Springer-Verlag, Berlin Heidelberg (1987), the entiredisclosure of which is incorporated herein by reference. The suitabilityof complexing agents for stabilizing the compositions can be determinedby one of ordinary skill in the art in the light of the presentdisclosure. In some embodiments, the cation-insulin compound conjugatecompositions specifically exclude or are substantially devoid of acomplexing agent.

A preferred complexing agent is protamine. In a solid form, theprotamine will preferably be present in about 3:1 to about 1:3 molarratio of insulin compound to protamine, more preferably about 2:1 toabout 1:2 molar ratio, ideally about 1:1 molar ratio. In someembodiments, the cation-insulin compound conjugate compositionsspecifically exclude or are substantially devoid of protamine.

Amino acids may also be used as complexing agents, e.g., glycine,alanine, valine, leucine, isoleucine, serine threonine, phenyl alanine,proline, tryptophan, asparagine, glutamic acid, and histidine, andoligopeptides, such as diglycine.

Carboxylic acids are also suitable for use as complexing agents;examples include acetic acid, and hydroxycarboxylic acids, such ascitric acid, 3-hydroxybutyric acid, and lactic acid.

7.6 Stabilizing Agents

In some embodiments, the cation-insulin compound conjugate compositionsinclude one or more stabilizing agents. Preferred stabilizing agentsinclude phenolic compounds and aromatic compounds. Preferred phenoliccompounds are phenol, m-cresol and m-paraben or mixtures thereof. Thestabilizing agent may be provided in any amount that improves stabilityof the cation-insulin compound conjugate compositions relative to ascientifically acceptable control, such as a correspondingcation-insulin compound conjugate composition in the absence of thestabilizing agent.

7.7 Presentation of Complexes

The complexes may be provided as a dry solid, such as a substantiallypure powder of cation-insulin compound conjugate, or a powder includinga cation-insulin compound conjugate solid along with otherpharmaceutically acceptable components. The complexes may also beprovided in a dissolved state in aqueous or organic medium, and/or asundissolved solids in such mediums.

7.7.1 Solid Compositions

The cation-insulin compound conjugate complexe may be provided as as asolid. The solid may, for example be in a dried state or in anundissolved state in an aqueous solution, organic solvent, emulsion,microemulsion, or oher non-dried form.

In one embodiment, the cation-insulin compound conjugate complexe isprovided as a pure processed solid composition. In a pure processedsolid composition, the molar ratio of insulin compound conjugate tocation is typically about 3:4 to about 3:0.5 (insulin compoundconjugate:cation), about 3:3.5 to about 3:1, or ideally about 3:1.

In a processed pure solid T-type composition (with cation, insulincompound conjugate and without protamine), the molar ratio of insulincompound conjugate to cation is typically about is typically about 3:4to about 3:0.5 (insulin compound conjugate:cation), about 3:3.5 to about3:1, or ideally about 3:1. In a processed pure solid T-type protaminecomposition (with cation, insulin compound conjugate and protamine), themolar ratio of insulin compound conjugate to cation is typically about3:6 to about 3:0.5 (insulin compound conjugate:cation), about 3:5 toabout 3:1, or ideally about 3:2.

In a processed pure solid R-type (lente) composition (with cation,insulin compound and stabilizing compound (e.g., a phenolic compound),and without protamine), the molar ratio of insulin compound conjugate tocation can typically range from about 3:4.5 to about 3:0.9, preferablyabout 3:3.9 to about 3:2.4. In a processed pure solid R-type(ultralente) composition (with cation, insulin compound and stabilizingcompound (e.g., a phenolic compound), and without protamine), the molarratio of insulin compound conjugate to cation can typically range fromabout 3:12 to greater than about 3:4.5, preferably about 3:9 to about3:4.8, more preferably about 3:6 to about 3:5.4. In a processed puresolid R-type protamine composition (with cation, insulin compound andstabilizing compound (e.g., a phenolic compound), and protamine), themolar ratio of insulin compound conjugate to cation can typically rangefrom about 3:12 to about 3:3, preferably about 3:9 to about 3:4.5, morepreferably about 3:6.9 to about 3:5.4.

For a monovalent cation, such as Na⁺, the solid would be expected tohave an insulin compound conjugate to cation ratio of about 3:6 to about3:3.

Solid compositions of the invention may, for example, includecompositions, such as powders, including insulin compound conjugatesand/or cation-insulin compound conjugate complexes of the invention.Preferably the solid compositions are provided at a pharmaceuticallyacceptable level of purity, i.e., free of contaminants which wouldunacceptably diminish the suitability of the compositions for use inhumans.

In some embodiments, compositions are provided in which thecation-insulin compound conjugate component is greater than about 90%crystalline, preferably greater than about 95% crystalline, morepreferably greater than about 99% crystalline. In other embodiments,compositions are provided in which the cation-insulin compound conjugatecomponent is greater than about 90% amorphous solids, preferably greaterthan about 95% amorphous solids, more preferably greater than about 99%amorphous solids.

In still other embodiments, compositions are provided in which thecation-insulin compound conjugate component is present in a mixture ofamorphous solids and crystalline solids. For example, the ratio ofamorphous solid to crystalline solid may be from about 1:10 to about10:1, or about 1:9 to about 9:1, or about 1:8 to about 8:1, or about 1:7to about 7:1, or about 1:6 to about 6:1, or about 1:5 to about 5:1, orabout 1:4 to about 4:1, or about 1:3 to about 3:1, or about 1:2 to about2:1, or about 1:1.

Furthermore, compositions can be provided using mixtures ofcation-insulin compound solids having different insulin compounds, suchas a solid including native insulin compound with a solid includinginsulin compound conjugates, or solids including one insulin compoundconjugate with a solid including a different insulin compound conjugate.

Moreover, the solid type and insulin compound/insulin compound conjugatecomponent may all vary. For example, compositions can be provided whichinclude Zn-insulin compound crystals using native insulin compound andamorphous insulin compound conjugates, or compositions can be providedwhich include amorphous Zn-insulin compound solids using native insulincompound and crystalline Zn-insulin compound conjugates. Such mixturesmay be used to achieve variations in physical characteristics, such asdissolution profile and/or variations in pharmacokinetic profile.

The average particle size of the solids are preferably in the range ofabout 0.1 to about 100 microns, more preferably 1-50 microns, still morepreferably 1-25 microns, ideally 1-15 microns. Small particle sizes maybe obtained by microcrystallization conditions, spray drying, milling,vacuum drying, freeze drying and the like.

In one embodiment the composition, when dried, contains greater thanabout 96% w/w insulin compound conjugate and from about 0.05, 0.1, 0.15,or 0.2 to about 4% w/w zinc. In another embodiment the composition, whendried, contains greater than about 91% w/w insulin compound conjugate,from about 0.05, 0.1, 0.15, or 0.2 to about 4% w/w zinc, and from about0.2 to about 5% w/w phenol. In yet another embodiment the composition,when dried, contains greater than about 82% w/w insulin compoundconjugate, from about 0.05, 0.1, 0.15, or 0.2 to about 4% w/w zinc, fromabout 0.2 to about 14% w/w protamine. In yet another embodiment thecomposition, when dried, contains greater than about 71% w/w insulincompound conjugate, from about 0.05, 0.1, 0.15, or 0.2 to about 4% w/wzinc, from about 0.2 to about 14% w/w protamine, and from about 0.2 toabout 15% w/w phenol.

In another embodiment the composition, when dried, includes from about0.1 to about 2% w/w Zn⁺⁺, and from about 0.08 to about 1% w/w phenol,preferably from about 0.5 to about 1.3% w/w Zn⁺⁺, andfrom about 0.1 toabout 0.7% w/w phenol, more preferably from greater than or equal to 1to about 3.5% w/w Zn⁺⁺, and from about 0.1 to about 3% w/w phenol, andstill more preferably from greater than or equal to 1.3 to about 2.2%w/w Zn⁺⁺, and from about 0.4 to about 2% w/w phenol.

The complexes can be provided in a lente-type preparation. For example,in a preferred dried lente-type preparation, Zn is provided in an amountranging from about 0.1 to about 2% w/w and phenol is present in anamount ranging from about 0.08 to about 1% w/w, with the remaining % w/wbeing insulin compound conjugate. Ideally, for a dried lente-typepreparation, Zn is provided in an amount ranging from about 0.5 to about1.3% w/w and phenol is present in an amount ranging from about 0.1 toabout 0.7% w/w, with the remaining % w/w being insulin compoundconjugate.

The complexes can be provided in an ultralente-type preparation. Forexample, in a preferred dried ultralente-type preparation, Zn isprovided in an amount ranging from greater than or equal to 1 to about3.5% w/w, and phenol is present in an amount ranging from about 0.1 toabout 3% w/w, with the remaining % w/w being insulin compound conjugate.Ideally, for a dried ultralente-type preparation, Zn is provided in anamount ranging from greater than or equal to 1.3 to about 2.2% w/w, andphenol is present in an amount ranging from about 0.4 to about 2% w/w,with the remaining % w/w being insulin compound conjugate.

7.7.2 Liquid Compositions

The cation-insulin compound conjugate complexes may be provided ascomponents undissolved components of a liquid. For example, the liquidmay be an aqueous solution including a cation-insulin compound conjugateas a precipitate, or the cation-insulin compound conjugate may beprovided as a component of a suspension, emulsion or microemulsion. Theliquid may also include dissolved components or complexes, along withthe undissolved components.

7.7.3 Mixtures and Co-Crystals

The compositions of the invention may, for example, include complexmixtures, solid mixtures, hybrid complexes and co-crystals.

Thus, for example, the invention provides compositions which include twoor more insulin compound conjugates and/or unconjugated insulincompounds. Further, where the compositions include solids, the solidsmay have different forms. Thus, for example, on solid may be crystallineand another solid may be an amorphous solid. As noted elsewhere, thesolids may be provided in a dried form or may be provided as solidcomponents of a liquid mixture. In a preferred embodiment, the mixtureof the invention includes two or more different insulin compoundconjugates, and the different insulin compound conjugates have differentsolubilities. In one embodiment, one of the complexes comprises alipophilic insulin compound conjugate and the other comprises ahydrophilic insulin compound conjugate. In still another embodiment, thecomplexes may include different insulin compound conjugates, where oneore more of the complexes has a circulation half-life of from about 1 toabout 4 hours, and one or more the complexes has a circulation half-lifethat is significantly greater than the circulation half-life of thefirst complex. In a related embodiment, one of the complexes has arapid-acting profile and another of the complexes has a medium-to-longacting profile. Furthermore, one of the complexes may have profilesuitable for basal insulin compound control while another has a profilesuitable for post-prandial glucose control. Preferred mixtures aremixtures of HIM2 and insulin, mixtures of HIM2 and IN105, mixtures ofIN105 and insulin compound, mixtures of IN105 and fatty acid acylatedinsulin, mixtures of HIM2 and fatty acid acylated insulin. Suitablefatty acid acylated insulins are described in the following U.S.patents, the entire disclosures of which are incorporated herein byreference: U.S. Pat. No. 6,531,448, entitled “Insoluble compositions forcontrolling blood glucose,” issued 11 Mar. 2003; U.S. Pat. No. RE37,971,entitled “Selective acylation of epsilon-amino groups,” issued 28 Jan.2003; U.S. Pat. No. 6,465,426, entitled “Insoluble insulincompositions,” issued 15 Oct. 2002; U.S. Pat. No. 6,444,641, entitled“Fatty acid-acylated insulin analogs.” issued 3 Sep. 2002; U.S. Pat. No.6,335,316, entitled “Method for administering acylated insulin,” issued1 Jan. 2002; U.S. Pat. No. 6,268,335, entitled “Insoluble insulincompositions,” issued 31 Jul. 2001; U.S. Pat. No. 6,051,551, entitled“Method for administering acylated insulin,” issued 18 Apr. 2000; U.S.Pat. No. 5,922,675, entitled “Acylated Insulin Analogs,” issued 13 Jul.1999; U.S. Pat. No. 5,700,904, entitled “Preparation of an acylatedprotein powder,” issued 23 Dec. 1997; U.S. Pat. No. 5,693,609, entitled“Acylated insulin analogs Granted,” issued 2 Dec. 1997; U.S. Pat. No.5,646,242, entitled “Selective acylation of epsilon-amino groups,” issue8 Jul. 1997; U.S. Pat. No. 5,631,347, entitled “Reducing gelation of afatty acid-acylated protein,” issued 20 May 1997; U.S. Pat. No.6,451,974, entitled “Method of acylating peptides and novel acylatingagents,” issued 17 Sep. 2002; U.S. Pat. No. 6,011,007, entitled“Acylated insulin,” issued 4 Jan. 2000; U.S. Pat. No. 5,750,497,entitled “Acylated insulin Granted: 12 May 1998; U.S. Pat. No.5,905,140, entitled “Selective acylation method,” issued May 18, 1999;U.S. Pat. No. 6,620,780, entitled “Insulin derivatives,” issued Sep. 16,2003; U.S. Pat. No. 6,251,856, entitled “Insulin derivatives,” issuedJun. 26, 2001; U.S. Pat. No. 6,211,144, entitled “Stable concentratedinsulin preparations for pulmonary delivery,” issued Apr. 3, 2001; U.S.Pat. No. 6,310,038, entitled “Pulmonary insulin crystals,” issued Oct.30, 2001; and U.S. Pat. No. 6,174,856, entitled “Stabilized insulincompositions,” issued Jan. 16, 2001. Especially preferred mono-fattyacid acylated insulins having 12, 13, 14, 15, or 16-carbon fatty acidscovalently bound to Lys(B29) of human insulin.

In one embodiment, the invention provides a co-crystal having twodifferent insulin compounds and/or insulin compound conjugates.Preferably the co-crystal exhibits one or more of the followingcharacteristics: substantially homogenous dissolution, a single in vivodissolution curve, and/or a single peak pharmacodynamic profile.Preferred co-crystals are co-crystals of HIM2 and insulin, co-crystalsof HIM2 and IN105, co-crystals of IN105 and insulin compound.

In one embodiment, the co-crystal includes human insulin, andco-crystallization with human insulin reduces the solubility of thecrystal relative to the solubility of a corresponding crystal of theinsulin compound conjugate. In another embodiment, the co-crystalincludes human insulin, and co-crystallization with human insulindecreases the solubility of the crystal relative to the solubility of acorresponding crystal of the insulin compound conjugate.

In another embodiment, the co-crystal includes a rapid acting, rapidclearing, and/or highly potent insulin compound conjugate, and along-acting, slow clearing, and/or poorly potent insulin compoundconjugate. Preferably the co-crystal has a PK/PD profile suitable forpost-prandial glucose control or for overnight basal insulin compoundcontrol.

In another embodiment, the invention provides a mixture or co-crystal inwhich an insulin compound conjugate is included with human insulin orlyspro insulin. The mixtures of the invention may include two differentinsulin compound conjugates. The mixtures may include an insulincompound conjugate and an unconjugated insulin compound. The mixturesmay include different insulin compound conjugates with different insulincompounds.

Further, the invention provides complexes having two different insulincompound conjugates and/or an insulin compound conjugate and anunconjugated insulin compound. The invention provides hybrid co-crystalsof two, three or more different insulin compound conjugates. Theinvention provides a complex having an insulin compound conjugate withan unconjugated insulin compound. The invention provides a co-crustalwith two or more different hydrophilic insulin compound conjugates; twoor more different hydrophobic insulin compound conjugates; two or moredifferent amphiphilic insulin compound conjugates; a hydrophilic insulincompound conjugate and a lipophilic insulin compound conjugate; ahydrophilic insulin compound conjugate and an unconjugated insulincompound; HIM2 together with an unconjugated insulin compound; IN105together with an unconjugated insulin compound; HIM2 together withIN105; HIM2 together with insulin compound and IN105; and othercombinations of the foregoing elements. As mentioned elsewhere, thecomplexes may be provided as dried solids, as dissolved complexes insolution and/or as undissolved complexes in solution. Variouscombinations may, for example, be employed to provide a complex orco-crystal having an extended profile.

7.8 Solubility of Complexes of the Invention

Preferably the aqueous solubility of the cation-insulin compoundconjugate complex at a pH of about 7.4 is from about 1/15, 1/14, 1/13,1/12, 1/11, 1/10, 1/9, 1/8, 1/7, 1/6, 1/5 up to about 0, 1, 2, 3, 4, 5,6, 7, 8, 9, or <10 times the aqueous solubility of the uncomplexedinsulin compound conjugate. Any combination of the foregoing upper andlower limits is within the scope of the invention. However, a preferredrange is from about 1/15 to <5, more preferred is about 1/10 to about 2,ideal is about 1/10 to <0. In a particularly surprising aspect of theinvention, the aqueous solubility of the cation-insulin compoundconjugate in solution at a pH of about 7.4 is often substantially lessthan the aqueous solubility of the insulin compound conjugate insolution at a pH of about 7.4. However, it will be appreciated that incertain embodiments, the aqueous solubility of the cation-insulincompound conjugate in solution at a pH of about 7.4 may be the same as,greater than, or substantially greater than, the aqueous solubility ofthe insulin compound conjugate in solution at a pH of about 7.4.

In one surprising embodiment, the aqueous solubility of thecation-insulin compound conjugate complex at a pH of about 7.4 issubstantially less than the solubility of the corresponding uncomplexedinsulin compound conjugate in solution at a pH of about 7.4, and thecation-insulin compound conjugate complex remains soluble at greaterthan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or 130 g/L inaqueous solution across a pH range beginning at about 5.5, 5.6, 5.7,5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.1, or 6.9 and endingat about 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, or 8.9. In yet another embodiment, the aqueous solubility of thecation-insulin compound conjugate complex at a pH of about 7.4 issubstantially less than the solubility of the corresponding insulincompound conjugate in solution at a pH of about 7.4, and thecation-insulin compound conjugate complex remains soluble at greaterthan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or 130 g/L inaqueous solution across a pH range from about 5.8 to about 8.5,preferably across a pH range from about 6.5 to about 8, more preferablyacross a pH range from about 6.9 to about 7.8.

Preferably the insulin compound conjugates of the invention are selectedto produce crystals in aqueous solution at a pH which is equal to pI +/−about 2.5, where the concentration of insulin compound conjugate is fromabout 0.5 mg/ml to about 50 mg/ml, preferably about 5 mg/ml to about 30mg/ml, more preferably about 15 mg/ml to about 30 mg/ml, and the crystalformulation begins to occur at about 3, 4 or 5% w/w/cation to insulincompound conjugate, where the cation is preferable Z⁺⁺. Preferablycrystals are present for a monoconjugate without protamine in an aqueoussolution at a pH ranging from about 4, 4.1, 4.2, 4.3 or 4.4 to about5.2, 5.3, 5.4, 5.5, 5.6, 5.7 or 5.8, preferably at pH of about 4 to<6.5, preferably about 4 to <5.8, preferably about 4.2 to about 5.5,more preferably about 4.4 to about 5.2. Preferably crystals are presentfor a diconjugate without protamine at pH of about 3.5 to <5.8,preferably about 3.8 to about 5.5, more preferably about 4.0 to about5.2. Preferably crystals are present for a triconjugate withoutprotamine at pH of about 3 to <5.5, preferably about 3.3 to about 5,more preferably about 3.8 to about 4.8.

7.8.1 R-Type Complexes

Preferably the aqueous solubility of the R type Zn complex of theinsulin compound conjugate at a pH of about 7.4 has a range of about 10to about 150 g/L, more preferably about 20 to about 130 g/L, morepreferably about 30 to about 110 g/L, more preferably about 35 to about60 g/L.

Preferably the aqueous solubility of the R type Zn complex of theinsulin compound conjugate with protamine at a pH of about 7.4 has arange of about 10 to about 110 g/L, more preferably about 20 to about 85g/L, more preferably about 30 to about 70 g/L.

7.8.2 T-Type Complexes

Preferably the aqueous solubility of the T-type Zn complex of theinsulin compound conjugate at a pH of about 7.4 has a range of about 30to about 175 g/L, more preferably about 50 to about 160 g/L, morepreferably about 70 to about 150 g/L.

Preferably the aqueous solubility of the T-type Zn complex of theinsulin compound conjugate with protamine at a pH of about 7.4 has arange of about 10 to about 150 g/L, more preferably about 20 to about130 g/L, more preferably about 30 to about 110 g/L, more preferablyabout 35 to about 60 g/L.

7.8.3 NPH-Type Complexes

Preferably the aqueous solubility of the NPH-type complex, of theinsulin compound conjugate at a pH of about 7.4 has a range of aboutabout 1 to about 150 g/L, more preferably about 5 to about 120 g/L,still more preferably about 10 to about 90 g/L.

7.9 Pharmaceutical Properties

Complexation of the insulin compound conjugate with cation generallyresults in improved pharmaceutical properties of the insulin compoundconjugate, relative to a scientifically acceptable control, such as acorresponding uncomplexed insulin compound conjugate.

In some cases, the complexed insulin compound conjugate will exhibit anextended or otherwise altered pK profile relative to a scientificallyacceptable control, such as a corresponding uncomplexed insulin compoundconjugate. In certain cases, the pK profile will exhibit a lispro-likeprofile. pK profile can be assessed using standard in vivo experiments,e.g., in mice, rats, dogs, or humans. Assays described herein forassessing the attributes of cation-insulin compound conjugate complexesare an aspect of the invention.

The complexes may exhibit improved chemical stability. Variousattributes of stability can be assessed by exposing the complex tovarious assay conditions such as the presence of plasma, the presence ofproteases, the presence of liver homogenate, the presence of acidicconditions, and the presence of basic conditions. Stability is improvedrelative to uncomplexed insulin compound conjugate when stability of thecomplexed insulin compound conjugate in any one or more of these assayconditions is greater than stability of the uncomplexed insulin compoundconjugate in the same conditions. A preferred assay for determiningstability in an acidic environment involves exposing the complexedinsulin compound conjugate to a solution having a pH of 2 for at least 2hours, where decreased degradation of the complexed insulin compoundconjugate relative to a scientifically acceptable control, such as acorresponding uncomplexed insulin compound conjugate, is indicative ofimproved stability. In vivo assays can also be used to test stability.For example, stability of the complexed insulin compound conjugate canbe tested by exposure to the gastrointestinal tract of a subject andcomparison with an appropriate control.

7.10 Method of Making

The invention also provides a method of making cation-insulin compoundconjugate compositions described herein. The method generally involvescontacting one or more insulin compound conjugates, as described herein,with one or more cations, as described herein, to form a solid.

For a divalent cation, such as Zn⁺⁺, the molar ratio of insulin compoundconjugate to cation used to make the composition in an aqueous solutionwith an insulin compound concentration ranging from about 2 mg/ml toabout 50 mg/ml can typically range from about 1:15 (insulin compoundconjugate:cation) to about 1:0.4, preferably about 1:9 to about 1:2.

To make T-type solid (with cation and insulin compound conjugate andwithout protamine) in the aqueous solution conditions described above,the molar ratio of insulin compound conjugate to cation is preferablyabout 1:1.5 to 1:3, ideally about 1:2. To make R-type solid (withcation, insulin compound and stabilizing compound (e.g., a phenoliccompound), and without protamine) in the aqueous solution conditionsdescribed above, the molar ratio of insulin compound conjugate to cationis preferably about 1:4 to 1:9, preferably about 1:7 to about 1:9ideally about 1:8.

To make T-type protamine solid (with cation and insulin compoundconjugate and protamine) in the aqueous solution conditions describedabove, the molar ratio of insulin compound conjugate to cation ispreferably about 1:1.5 to 1:9, ideally about 1:2. To make R-typeprotamine solid (with cation, insulin compound and stabilizing compound(e.g., a phenolic compound), and protamine) in the aqueous solutionconditions described above, the molar ratio of insulin compoundconjugate to cation is preferably about 1:2 to 1:15, preferably about1:7 to about 1:9 ideally about 1:8.

The insulin compound conjugate is preferably added to the buffer in anamount which is calculated to achieve a concentration in the range offrom greater than 2 to about 100 g/L, preferably from about 3, 4, 5, 6,7, 8, 9 or 10 to about 40 g/L, more preferably from about 10 to about 30g/L.

Where the cation is divalent (e.g., Zn⁺⁺, Ca⁺⁺), it is preferably addedin an amount which calculated to achieve a concentration in the range offrom about 0.04 to about 10 g/L, preferably from about 0.1 to about 5g/L, more preferably from about 0.2 to about 4 g/L. For T-type crystalsor T-type protamine crystals, the cation concentration is preferably inthe range of from about 0.04 to about 1 g/L, more preferably about 0.1to about 0.3 g/L. For R-type crystals or R-type protamine crystals, thecation concentration is preferably in the range of from about 1 to about5 g/L, more preferably about 1.5 to about 4 g/L.

Where the cation is monovalent, it is preferably added in an amountwhich calculated to achieve a concentration in the range of from about0.08 to about 40 g/L, preferably from about 0.4 to about 20 g/L, morepreferably from about 0.8 to about 16 g/L.

The method may further include combining a stabilizing agent with thecation and insulin compound conjugate. Preferred stabilizing agents aredescribed above. When used, the stabilizing agent is added in an amountsufficient to provide a greater degree of solid formation than isachieved using the same reagents and reaction conditions in the absenceof the stabilizing agent. Where the stabilizing agent is a phenoliccompound (e.g., phenol, m-cresol, m-paraben), can be added in an amountranging from about 10 to about 50% w/w, more preferably from about 20 toabout 40% w/w, still more preferably from about 25 to about 35% w/w. Ina more preferred embodiment, the stabilizing agent is a phenoliccompound (e.g., phenol, m-cresol, m-paraben), can be added in an amountranging from about 0.01 to about 10% w/w, more preferably 0.01 to about5% w/w, still more preferably 0.01 to about 1% w/w. Thus, in oneembodiment, the method involves combining insulin compound conjugate, acation and a stabilizing agent in an aqueous solution to yield thecation-insulin compound conjugate composition, where the combination mayyield solubilized complexes and/or crystalline or non-crystallinesolids.

The method may further include the use of a complexing agent, such asprotamine, which is combined with the cation and insulin compoundconjugate, and optionally also includes a stabilizing agent.

To prepare a solid in an aqueous solution having a pH in the range ofabout 5 to about 8, protamine is preferably provided in an amountrelative to insulin compound conjugate of about 4 to about 45% w/w(protamine/insulin compound), preferably about 8 to about 25% w/w, morepreferably about 9 to about 20% w/w, ideally about 10 to about 12% w/w.For T-type solids, a preferred pH range is from about 5 to about 6, morepreferably about 5 to about 5.5, still more preferably about 5.1 toabout 5.3, ideally about 5.2. For R-type solids, a preferred pH range isfrom about 6 to about 7, more preferably about 6.2 to about 6.8, stillmore preferably about 6.4 to about 6.6, ideally about 6.5.

The inventors have surprisingly discovered that T-type complexes can beconverted to protamine T-type complexes in the absence of a stabilizingagent, such as phenol. The T-type complex is made by complexing Zn withthe insulin compound molecule in aqueous solution in the absence ofphenol. Protamine is then added to convert the T-type complex into aprotamine T-type complex. Amounts and pH ranges are as described above.

Thus, in one embodiment, the method involves combining insulin compoundconjugate, a cation and a complexing agent in an aqueous solution toyield the cation-insulin compound conjugate composition, where thecombination may yield solubilized complexes and/or crystalline oramorphous solids. In another embodiment, the method involves combininginsulin compound conjugate, a cation, a complexing agent, and astabilizing agent in an aqueous solution to yield the cation-insulincompound conjugate composition, where the combination may yieldsolubilized complexes and/or crystalline or amorphous solids.

In some embodiments, the compositions can include preservatives.Examples of suitable preservatives include benzyl alcohol,p-hydroxybenzoic acid esters, glycerol. Stabilizing agents, such asphenol, m-cresol, and m-paraben, can also be used as preservatives.Glycerol and phenol are suitably added together to enhance antimicrobialeffectiveness.

Other components useful in preparing the solids include isotonic agents,such as NaCl, glycerol, and monosaccharides.

The cation insulin compound conjugate solids can typically be formedrelatively quickly. For example, solid formation is typically completewithin three days, often within 24 hours. It may be desirable in someinstances to slow the reaction down in order to improve crystalformation.

In one embodiment of the invention, the solids are formed at roomtemperature (25° C.) without requiring temperature reduction forinducing precipitation of solids. For example, room temperature iseffective for R-type and T-type crystals. The temperature for solidformation is preferably about 0 to about 40° C., preferably about 17 toabout 30° C., and more preferably about 22 to about 27° C., ideallyabout 25° C.

In one embodiment, the method includes combining in an aqueous solutionan insulin compound conjugate and a metal cation to provide acrystalline or amorphous solid. The aqueous solution containing theinsulin compound conjugate to which the cation will be added ispreferably a buffered solution having a pH in the range of pI of theinsulin compound conjugate +/− about 1.5, preferably pI +/− about 1,more preferably pI +/− about 0.75. These ranges also apply to T-type,R-type and protamine complexes. However, for neutral protamine complexes(NPH-type), the preferred pH is about 7 to about 8.5, more preferablyabout 7.5 to about 8. Once the metal cation is added, the pH may changeslightly, and the pH may be adjusted to target the pH ranges describedabove. With phenolic compounds, there may be a minor pH change, and anacid or base can be used to adjust to the preferred ranges.

pI values for insulin compound conjugates typically require a pH of lessthan about 7, preferably less than about 6, more preferably less thanabout 5.5. Human insulin monoconjugates with neutral modifying moietiestypically have a pI range of about 4.75+/−0.25. For human insulindiconjugates, the pI range is typically 4.25+/−0.25. For human insulintriconjugates, the pI range is typically 3.5+/−0.25.

Examples of suitable buffer systems include ammonium acetate buffer,sodium phosphate buffer, tris buffer, mixture of sodium phosphate andammonium acetate, sodium acetate buffer, mixture of sodium acetate andammonium acetate, and citric acid buffer, and any of the foregoingbuffer systems [A] also containing ethanol and/or acetonitrile [B](e.g., at percent ratio of A:B of about 1:1 to about 10:1). It is asurprising aspect of the invention that the cation-insulin compoundconjugate solid can be formed in an aqueous mixture containing anorganic solvent, such as ethanol or acetonitrile.

One unique feature of the invention is that in addition to providinguseful cation-insulin compound conjugate complexes, the invention alsoprovides a method of separating cation-insulin compound conjugates fromunconjugated insulin compound in the manufacturing process. In thisprocess, the cation-insulin compound conjugates can be precipitated outof solution and the solubilized unconjugated insulin compound can beremoved by filtration, for example. This feature eliminates 2 steps inthe manufacture of insulin compound conjugates: the concentration stepand the lyophilization step.

Processed pure solid composition may be formed using standardtechniques, such as centrifugation and/or filtration, followed bywashing (e.g., with ethanol/water), and lyophilization or vacuum drying.Multiple washings may be used to adjust phenol and/or cation content.

7.11 Formulation

The complexes may be formulated for administration in a pharmaceuticalcarrier in accordance with known techniques. See, e.g., Alfonso R.Gennaro, Remington: The Science and Practice of Pharmacy, LippincottWilliams & Wilkins Publishers (June 2003), and Howard C. Ansel,Pharmaceutical Dosage Forms and Drug Delivery Systems, LippincottWilliams & Wilkins Publishers, 7th ed. (October 1999), the entiredisclosures of which are incorporated herein by reference for theirteachings concerning the selection, making and using of pharmaceuticaldosage forms.

The complexes, typically in the form of an amorphous or crystallinesolid, can be combined with a pharmaceutically acceptable carrier. Thecarrier must be acceptable in the sense of being compatible with anyother ingredients in the pharmaceutical composition and should not beunduly deleterious to the subject, relative to the benefit provided bythe active ingredient(s). The carrier may be a solid or a liquid, orboth. It is preferably formulated as a unit-dose formulation, forexample, a tablet. The unit dosage form may, for example, contain fromabout 0.01 or 0.5% to about 95% or 99% by weight of the cation-insulincompound complex. The pharmaceutical compositions may be prepared by anyof the well known techniques of pharmacy including, but not limited to,admixing the components, optionally including one or more accessoryingredients.

Examples of suitable pharmaceutical compositions include those made fororal, rectal, inhalation (e.g., via an aerosol) buccal (e.g.,sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular,intradermal, intraarticular, intrapleural, intraperitoneal,inracerebral, intraarterial, or intravenous), topical, mucosal surfaces(including airway surfaces), nasal surfaces, and transdermaladministration. The most suitable route in any given case will depend onthe nature and severity of the condition being treated and on the natureof the particular cation-insulin compound complexes being used.Preferred oral compositions are compositions prepared for ingestion bythe subject. Ideally, the oral compositions are prepared to survive orsubstantially survive passage through the stomach and to completely orsubstantially completely dissolve in the intestine for delivery of theactive ingredient. Examples of suitable transdermal systems includeultrasonic, iontophoretic, and patch delivery systems.

In one aspect, the invention provides fatty acid compositions comprisingone or more saturated or unsaturated C₄, C₅, C₆, C₇, C₈, C₉ or C₁₀ fattyacids and/or salts of such fatty acids. Preferred fatty acids arecaprylic, capric, myristic and lauric. Preferred fatty acid salts aresodium salts of caprylic, capric, myristic and lauric acid.

Preferred fatty acid compositions include a single fatty acid or asingle fatty acid salt and do not include substantial amounts of otherfatty acids or fatty acid salts. In one aspect of the invention, thefatty acid content of the composition is greater than about 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, or 99.9% w/w asingle fatty acid. In another embodiment, the fatty acid content of thecomposition is within a range having as a lower limit of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0%w/w, and having as an upper limit of about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3,6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4,10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6,11.7, 11.8, 11.9, or 12.0% w/w. In yet another embodiment, the fattyacid content of the composition is within a range having as a lowerlimit about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, or 3.0% w/w, and having as an upper limit about 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1,10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3,11.4, 11.5, 11.6, 11.7, 11.8, 11.9, or 12.0% w/w, and the fatty acidcontent of the composition is greater than about 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, or 99.9% w/w a single fattyacid.

Active components of these formulations may include conjugated orunconjugated, complexed or uncomplexed proteins and/or peptides.Preferred proteins and/or peptides are those described herein. Preferredconjugates are those described herein. Preferred complexes are thosedescribed herein. Preferred oral compositions are compositions preparedfor ingestion by the subject. Ideally, the oral compositions areprepared to survive or substantially survive passage through the stomachand to completely or substantially completely dissolve in the intestinefor delivery of the active ingredient. The formulation may in some casesinclude an enteric coating, and in some cases, the formulation willspecifically exclude an enteric coating. The composition is preferablyprovided as a tablet, powder, hard gelatin capsule, or soft gelatincapsule, though other forms described herein are suitable as well.

The fatty acid compositions of the invention may include fatty acidacylated insulins. Examples of suitable fatty acid acylated insulins aredescribed in the following U.S. patents, the entire disclosures of whichare incorporated herein by reference: U.S. Pat. No. 6,531,448, entitled“Insoluble compositions for controlling blood glucose,” issued 11 Mar.2003; U.S. Pat. No. RE37,971, entitled “Selective acylation ofepsilon-amino groups,” issued 28 Jan. 2003; U.S. Pat. No. 6,465,426,entitled “Insoluble insulin compositions,” issued 15 Oct. 2002; U.S.Pat. No. 6,444,641, entitled “Fatty acid-acylated insulin analogs.”issued 3 Sep. 2002; U.S. Pat. No. 6,335,316, entitled “Method foradministering acylated insulin,” issued 1 Jan. 2002; U.S. Pat. No.6,268,335, entitled “Insoluble insulin compositions,” issued 31 Jul.2001; U.S. Pat. No. 6,051,551, entitled “Method for administeringacylated insulin,” issued 18 Apr. 2000; U.S. Pat. No. 5,922,675,entitled “Acylated Insulin Analogs,” issued 13 Jul. 1999; U.S. Pat. No.5,700,904, entitled “Preparation of an acylated protein powder,” issued23 Dec. 1997; U.S. Pat. No. 5,693,609, entitled “Acylated insulinanalogs Granted,” issued 2 Dec. 1997; U.S. Pat. No. 5,646,242, entitled“Selective acylation of epsilon-amino groups,” issue 8 Jul. 1997; U.S.Pat. No. 5,631,347, entitled “Reducing gelation of a fatty acid-acylatedprotein,” issued 20 May 1997; U.S. Pat. No. 6,451,974, entitled “Methodof acylating peptides and novel acylating agents,” issued 17 Sep. 2002;U.S. Pat. No. 6,011,007, entitled “Acylated insulin,” issued 4 Jan.2000; U.S. Pat. No. 5,750,497, entitled “Acylated insulin Granted: 12May 1998; U.S. Pat. No. 5,905,140, entitled “Selective acylationmethod,” issued May 18, 1999; U.S. Pat. No. 6,620,780, entitled “Insulinderivatives,” issued Sep. 16, 2003; U.S. Pat. No. 6,251,856, entitled“Insulin derivatives,” issued Jun. 26, 2001; U.S. Pat. No. 6,211,144,entitled “Stable concentrated insulin preparations for pulmonarydelivery,” issued Apr. 3, 2001; U.S. Pat. No. 6,310,038, entitled“Pulmonary insulin crystals,” issued Oct. 30, 2001; and U.S. Pat. No.6,174,856, entitled “Stabilized insulin compositions,” issued Jan. 16,2001. Especially preferred are mono-fatty acid acylated insulins having12, 13, 14, 15, or 16-carbon fatty acids covalently bound to Lys(B29) ofhuman insulin.

Pharmaceutical compositions suitable for oral administration may bepresented in discrete units, such as capsules, cachets, lozenges, ortables, each containing a predetermined amount of the mixture of insulincompound conjugates; as a powder or granules; as a solution or asuspension in an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil emulsion. Such formulations may be prepared by any suitablemethod of pharmacy which includes the step of bringing into associationthe mixture of insulin compound conjugates and a suitable carrier (whichmay contain one or more accessory ingredients as noted above).Formulations may include suspensions of solids, complexed cation-insulincompound conjugates, uncomplexed active ingredient (e.g., native insulincompound, insulin compound conjugates), and mixtures of the foregoing.

In general, the pharmaceutical compositions of the invention areprepared by uniformly and intimately admixing the complexes with aliquid or solid carrier, or both, and then, if necessary, shaping theresulting mixture. For example, a tablet may be prepared by compressingor molding a powder or granules containing the mixture of insulincompound conjugates, optionally with one or more accessory ingredients.Compressed tablets may be prepared by compressing, in a suitablemachine, the mixture in a free-flowing form, such as a powder orgranules optionally mixed with a binder, lubricant, inert diluent,and/or surface active/dispersing agent(s). Molded tablets may be made bymolding, in a suitable machine, the powdered composition moistened withan inert liquid binder.

Pharmaceutical compositions suitable for buccal (sub-lingual)administration include lozenges comprising the mixture of insulincompound conjugates in a flavoured base, usually sucrose and acacia ortragacanth; and pastilles comprising the mixture of insulin compoundconjugates in an inert base such as gelatin and glycerin or sucrose andacacia. Examples of suitable formulations can be found in U.S. PatentPublication Nos. 20030229022 (“Pharmaceutical formulation”); 20030236192(“Method of modifying the release profile of sustained releasecompositions”); 20030096011 (“Method of producing submicron particles ofa labile agent and use thereof”); 20020037309 (“Process for thepreparation of polymer-based sustained release compositions”);20030118660 (“Residual solvent extraction method and microparticlesproduced thereby”); as well as U.S. Pat. No. 6,180,141 (“Composite gelmicroparticles as active principle carriers”); U.S. Pat. No. 6,737,045(“Methods and compositions for the pulmonary delivery insulincompound”); U.S. Pat. No. 6,730,334 (“Multi-arm block copolymers as drugdelivery vehicles”); U.S. Pat. No. 6,685,967 (“Methods and compositionsfor pulmonary delivery of insulin compound”); U.S. Pat. No. 6,630,169(“Particulate delivery systems and methods of use”); U.S. Pat. No.6,589,560 (“Stable glassy state powder formulations; U.S. Pat. No.6,592,904 (“Dispersible macromolecule compositions and methods for theirpreparation and use”); U.S. Pat. No. 6,582,728 (“Spray drying ofmacromolecules to produce inhaleable dry powders”); U.S. Pat. No.6,565,885 (“Methods of spray drying pharmaceutical compositions”); U.S.Pat. No. 6,546,929 (“Dry powder dispersing apparatus and methods fortheir use”); U.S. Pat. No. 6,543,448 (“Apparatus and methods fordispersing dry powder medicaments”); U.S. Pat. No. 6,518,239 (“Drypowder compositions having improved dispersivity”); U.S. Pat. No.6,514,496 (“Dispersible antibody compositions and methods for theirpreparation and use”); U.S. Pat. No. 6,509,006 (“Devices compositionsand methods for the pulmonary delivery of aerosolized medicaments”);U.S. Pat. No. 6,433,040 (“Stabilized bioactive preparations and methodsof use”); U.S. Pat. No. 6,423,344 (“Dispersible macromoleculecompositions and methods for their preparation and use”); U.S. Pat. No.6,372,258 (“Methods of spray-drying a drug and a hydrophobic aminoacid”); U.S. Pat. No. 6,309,671 (“Stable glassy state powderformulations”); U.S. Pat. No. 6,309,623 (“Stabilized preparations foruse in metered dose inhalers”); U.S. Pat. No. 6,294,204 (“Method ofproducing morphologically uniform microcapsules and microcapsulesproduced by this method”); U.S. Pat. No. 6,267,155 (“Powder fillingsystems, apparatus and methods”); U.S. Pat. No. 6,258,341 (“Stableglassy state powder formulations”); U.S. Pat. No. 6,182,712 (“Powerfilling apparatus and methods for their use”); U.S. Pat. No. 6,165,463(“Dispersible antibody compositions and methods for their preparationand use”); U.S. Pat. No. 6,138,668 (“Method and device for deliveringaerosolized medicaments”); U.S. Pat. No. 6,103,270 (“Methods and systemfor processing dispersible fine powders”); U.S. Pat. No. 6,089,228(“Apparatus and methods for dispersing dry powder medicaments”); U.S.Pat. No. 6,080,721 (“Pulmonary delivery of active fragments ofparathyroid hormone”); U.S. Pat. No. 6,051,256 (“Dispersiblemacromolecule compositions and methods for their preparation and use”);U.S. Pat. No. 6,019,968 (“Dispersible antibody compositions and methodsfor their preparation and use”); U.S. Pat. No. 5,997,848 (“Methods andcompositions for pulmonary delivery of insulin compound”); U.S. Pat. No.5,993,783 (“Method and apparatus for pulmonary administration of drypowder.alpha.1-antitrypsin”); U.S. Pat. No. 5,922,354 (“Methods andsystem for processing dispersible fine powders”); U.S. Pat. No.5,826,633 (“Powder filling systems, apparatus and methods”); U.S. Pat.No. 5,814,607 (“Pulmonary delivery of active fragments of parathyroidhormone”); U.S. Pat. No. 5,785,049 (“Method and apparatus for dispersionof dry powder medicaments”); U.S. Pat. No. 5,780,014 (“Method andapparatus for pulmonary administration of dry powder alpha1-antitrypsin”); U.S. Pat. No. 5,775,320 (“Method and device fordelivering aerosolized medicaments”); U.S. Pat. No. 5,740,794(“Apparatus and methods for dispersing dry powder medicaments”); U.S.Pat. No. 5,654,007 (“Methods and system for processing dispersible finepowders”); U.S. Pat. No. 5,607,915 (“Pulmonary delivery of activefragments of parathyroid hormone”); U.S. Pat. No. 5,458,135 (“Method anddevice for delivering aerosolized medicaments”); U.S. Pat. No. 6,602,952(“Hydrogels derived from chitosan and poly(ethylene glycol) or relatedpolymers”); and U.S. Pat. No. 5,932,462 (“Multiarmed, monofunctional,polymer for coupling to molecules and surfaces”). Further, Suitablesustained release formulations are described in Cardinal Health's U.S.Pat. No. 5,968,554, entitled “A sustained release pharmaceuticalpreparation,” issued 19 Oct. 1999, the entire disclosure of which isincorporated herein by reference. Suitable microparticle formulationsare described in Spherics, Inc.'s International Patent PublicationWO/2003-049,701, entitled “Methods and products useful in the formationand isolation of microparticles,” published 30 Oct. 2003. Suitablebioadhesive formulations are described in Spherics, Inc.'s InternationalPatent Publication WO/2003-051,304, entitled “Bioadhesive drug deliverysystem with enhanced gastric retention”, published 6 May 2004.

Pharmaceutical compositions according to embodiments of the inventionsuitable for parenteral administration comprise sterile aqueous andnon-aqueous injection solutions of the complexes, which preparations arepreferably isotonic with the blood of the intended recipient. Thesepreparations may contain anti-oxidants, buffers, bacteriostats andsolutes which render the composition isotonic with the blood of theintended recipient. Aqueous and non-aqueous sterile suspensions mayinclude suspending agents and thickening agents. The compositions may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, an injectable, stable, sterile composition with a mixtureof complexes in a unit dosage form in a sealed container may beprovided. The mixture of complexes can be provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection into a subject. The parenteral unit dosage formtypically comprises from about 1 microgram to about 10 mg of the mixtureof complexes. When the complexes are substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecomplexes in an aqueous carrier. One such useful emulsifying agent isphosphatidyl choline.

A solid dosage form for oral administration typically includes fromabout 2 mg to about 500 mg, preferably about 10 mg to about 250 mg,ideally about 20 mg to about 110 mg of the complexes. Pharmaceuticalcompositions suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing thecomplexes with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Pharmaceutical compositions suitable for topical application to the skinpreferably take the form of an ointment, cream, lotion, paste, gel,spray, aerosol, or oil. Carriers which may be used include petroleumjelly, lanoline, PEGs, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Pharmaceutical compositions suitable for transdermal administration maybe presented as discrete patches adapted to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time.Compositions suitable for transdermal administration may also bedelivered by iontophoresis (see, for example, Pharmaceutical Research 3(6):318 (1986)) and typically take the form of an optionally bufferedaqueous solution of the mixture of insulin compound conjugates. Suitableformulations comprise citrate or bis/tris buffer (pH 6) or ethanol/waterand contain from 0.1 to 0.2M active ingredient.

In a preferred embodiment, the complexes are administered as componentsof solid fatty acid formulations as described in U.S. Patent ApplicationNo. 60/494,821, filed on 13 Aug. 2003, by Opawale et al., the entiredisclosure of which is incorporated herein by reference.

In certain embodiments, the insulin compound conjugate may be providedseparately from the cation and/or other components needed to form thesolids. For example, the insulin compound conjugate may be provided as adried solid, and the buffer solution including the cation, stabilizingagent, preservative and/or other component may be provided separately,so that the user may combine the separate components to produce thecation-insulin compound conjugate complexes.

7.12 Methods of Treatment

The cation-insulin compound conjugate compositions and formulationsthereof are useful in the treatment of conditions in which increasingthe amount of circulating insulin compound (relative to the amountprovided by the subject in the absence of administration of insulincompound from an exogenous source) yields a desirable therapeutic orphysiological effect. For example, the condition treated may be Type IoR-type II diabetes, prediabetes and/or metabolic syndrome. In oneembodiment, the compositions are administered to alleviate symptoms ofdiabetes. In another embodiment, the compositions are administered to aprediabetic subject in order to prevent or delay the onset of diabetes.

The effective amount of the cation-insulin compound conjugatecomposition for administration according to the methods of the inventionwill vary somewhat from mixture to mixture, and subject to subject, andwill depend upon factors such as the age and condition of the subject,the route of delivery and the condition being treated. Such dosages canbe determined in accordance with routine pharmacological proceduresknown to those skilled in the art.

As a general proposition, an oral dosage from about 0.025 to about 10mg/kg of active ingredient (i.e., the conjugate) will have therapeuticefficacy, with all weights being calculated based upon the weight of themixture of insulin compound conjugates. A more preferred range is about0.06 to about 1 mg/kg, and an even more preferred range is about 0.125to about 0.5 mg/kg

A parenteral dosage typically ranges from about 0.5 μg/kg to about 0.5mg/kg, with all weights being calculated based upon the weight of themixture of insulin compound conjugates. A more preferred range is about1 μg/kg to about 100 μg/kg.

The frequency of administration is usually one, two, or three times perday or as necessary to control the condition. Alternatively, thecation-insulin compound conjugate compositions may be administered bycontinuous infusion. The duration of treatment depends on the type ofinsulin compound deficiency being treated and may be for as long as thelife of the subject. The complexes may, for example, be administeredwithin 0 to 30 minutes prior to a meal. The complexes may, for example,be administered within 0 to 2 hours prior to bedtime.

8 SYNTHESIS EXAMPLES

The following examples are presented to illustrate and explain theinvention.

8.1 Synthesis of Protected MPEG₆C₃ oligomer(3-{2-[2-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionicacid tert-butyl ester)

Methyl hexaethylene glycol (1.0 g, 3.37 mmol) and tert-butyl acrylate(0.216 g, 1.69 mmol) were dissolved in dry THF (10 mL). Sodium metal 0.4mg, 0.016 mmol) was added to the solution. After stirring for 4 h atroom temperature, the reaction mixture was quenched by the addition of 1M HCl (15 mL). The quenched reaction mixture was then extracted withCH₂Cl₂ (1×50 mL, 1×25 mL). The organic layer was dried (MgSO₄) andconcentrated. After purification by silica gel chromatography (ethylacetate as eluent), the product was obtained as an oil (0.832 g, 58%).

8.2 Synthesis of the MPEG₆C₃ oligomer acid(3-{2-[2-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionicacid)

The tert-butyl ester (0.165 g, 0.0389 mmol) was deprotected by stirringat room temperature in trifluoroacetic acid (2.0 mL). The contents werethen concentrated to a constant weight (0.125 g, 87%).

8.3 Synthesis of the Activated MPEG₆C₃ oligomer(3-{2-[2-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionicacid 2,5-dioxo-pyrrolidin-1-yl ester)

The acid (0.660 g, 1.79 mmol) and N-hydroxysuccinimide (0.2278 g, 1.97mmol) were dissolved in dry CH₂Cl₂ (15 mL). Ethyl dimethylaminopropylcarbodiimide hydrochloride (EDC, 0.343 g, 1.79 mmol) was added. Afterstirring at room temperature overnight, the reaction mixture was dilutedwith CH₂Cl₂ and was washed with water (2×45 mL). The organic layer wasdried (MgSO₄) and concentrated to a constant weight. The product was anoil (0.441 g, 53%).

8.4 Synthesis of the Protected MPEG₄C₃ oligomer(3-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acidtert-butyl ester)

Methyl tetraethylene glycol (1.0 g, 4.80 mmol) and tert-butyl acrylate(0.308 g, 2.40 mmol) were dissolved in dry THF (10 mL). Sodium metal 0.6mg, 0.024 mmol) was added to the solution. After stirring for 4 h atroom temperature, the reaction mixture was quenched by the addition of 1M HCl (15 mL). The quenched reaction mixture was then extracted withCH₂Cl₂ (1×50 mL, 1×25 mL). The organic layer was dried (MgSO₄) andconcentrated. After purification by silica gel chromatography (ethylacetate as eluent), the product was obtained as an oil (1.28 g, 79%).

8.5 Synthesis of the MPEG₆C₃ oligomer acid(3-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid)

The tert-butyl ester (1 g, 3.42 mmol) was deprotected by stirring atroom temperature in trifluoroacetic acid (6.0 mL). The contents werethen concentrated to a constant weight (0.87 g, 91%).

8.6 Synthesis of the Activated MPEG₄C₃ oligomer(3-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid2,5-dioxo-pyrrolidin-1-yl ester)

The acid (0.6 g, 2.14 mmol) and N-hydroxysuccinimide (0.271 g, 2.35mmol) were dissolved in dry CH₂Cl₂ (20 mL). Ethyl dimethylaminopropylcarbodiimide hydrochloride (EDC, 0.409 g, 2.14 mmol) was added. Afterstirring at room temperature overnight, the reaction mixture was dilutedwith CH₂Cl₂ and was washed with water (2×45 mL). The organic layer wasdried (MgSO₄) and concentrated to a constant weight. The product was anoil (0.563 g, 69%).

8.7 Synthesis of the Protected MPEG₄C₃ oligomer(3-(2-Methoxy-ethoxy)-propionic acid tert-butyl ester)

Methyl tetraethylene glycol (5.0 g, 41.6 mmol) and tert-butyl acrylate(2.66 g, 20.8 mmol) were dissolved in dry THF (20 mL). Sodium metal 0.47mg, 20.8 mmol) was added to the solution. After stirring for 4 h at roomtemperature, the reaction mixture was quenched by the addition of 1 MHCl (30 mL). The quenched reaction mixture was then extracted withCH₂Cl₂ (1×100 mL, 1×50 mL). The organic layer was dried (MgSO₄) andconcentrated. After purification by silica gel chromatography (ethylacetate as eluent), the product was obtained as an oil (7.5 g, 89%).

8.8 Synthesis of the MPEG₆C₃ oligomer acid(3-[2-(2-Methoxy-ethoxy)-ethoxy]-propionic acid)

The tert-butyl ester (1 g, 4.90 mmol) was deprotected by stirring atroom temperature in trifluoroacetic acid (6.0 mL). The contents werethen concentrated to a constant weight (0.652 g, 89%).

8.9 Synthesis of 2-[2-(2-Propoxy-ethoxy)-ethoxy]-ethanol (1)

Triethylene glycol (19.5 g, 0.13 mol) was dissolved in tetrahydrofuran(150 mL) and sodium hydride (2.60 g, 0.065 mol) was added portion wiseover 0.5 h and the reaction was stirred for an additional 1 h. Then1-bromopropanol (8.0 g, 0.065 mol) dissolved in tetrahydrofuran (30 mL)was added dropwise via addition funnel and the reaction was stirredovernight at room temperature. Crude reaction mixture was filteredthrough Celite, washed CH₂Cl₂, and evaporated to dryness. The resultantoil was dissolved in CH₂Cl₂ (250 mL), washed sat. NaCl (250 mL), H₂O(250 mL), dried MgSO₄, and evaporated to dryness. Column chromatography(Silica, ethyl acetate) afforded 1 a yellowish oil (2.24 g, 18% yield).

8.10 Synthesis of carbonic acid 4-nitro-phenyl ester2-[2-(2-propoxy-ethoxy)-ethoxy]-ethyl ester

4-Nitrochloroformate (3.45 g, 17.1 mmol) and 1 (2.2 g, 11.4 mmol) weredissolved in CH₂Cl₂ (20 mL). After stirring for 10 min, TEA (2.1 mL, 15mmol) was added and reaction stirred overnight at room temperature.Crude reaction was diluted with CH₂Cl₂ (50 mL), washed 1M HCl (50 mL),H₂O (50 mL), dried MgSO₄, and evaporated to dryness. Columnchromatography (silica, ethyl acetate/hexanes, 3:2) afforded 2 ayellowish oil (2.57 g, 63% yield).

8.11 Synthesis of carbonic acid 2,5-dioxo-pyrrolidin-1-yl ester2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester

Triethylene glycol monomethyl ether (1.0 g, 6.1 mmol) andN,N′-disuccinimidyl carbonate (1.87 g, 7.3 mmol) were dissolved inacetonitrile (10 mL). Then triethylamine (1.3 mL, 9.15 mmol) was addedand the reaction stirred overnight at room temperature. Crude reactionwas evaporated to dryness, dissolved in sat. NaHCO₃ (50 mL), washedethyl acetate (2×50 mL), dried MgSO₄, and evaporated to dryness. Columnchromatography (Silica, ethyl acetate) afforded 1 a clear oil (0.367 g,20% yield).

8.12 Synthesis of carbonic acid 2,5-dioxo-pyrrolidin-1-yl ester2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethyl ester (1)

Tetraethylene glycol monomethyl ether (1.0 g, 4.8 mmol) andN,N′-disuccinimidyl carbonate (1.48 g, 5.8 mmol) were dissolved inacetonitrile (10 mL). Then triethylamine (1.0 mL, 7.2 mmol) was addedand the reaction stirred overnight at room temperature. Crude reactionwas evaporated to dryness, dissolved in sat. NaHCO₃ (30 mL), washedethyl acetate (2×30 mL), dried MgSO₄, and evaporated to dryness. Columnchromatography (Silica, ethyl acetate/MeOH, 20:1) afforded 1 a clear oil(0.462 g, 28% yield).

8.13 Synthesis of But-3-enoic acid ethyl ester

Vinylacetic acid (10.0 g, 0.12 mol) was dissolved in ethanol (200 mL)and conc. sulfuric acid (0.75 mL, 0.014 mol) was added. The reaction washeated to reflux for 4 h. Crude reaction was diluted with ethyl acetate(200 mL), washed H₂O (200 mL), sat. NaHCO₃ (200 mL), dried MgSO₄, andevaporated to dryness to afford 1 a clear oil (3.17 g, 23%).

8.14 Synthesis of 4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyricacid ethyl ester

Triethylene glycol monomethyl ether (4.27 g, 0.026 mol) and But-3-enoicacid ethyl ester (1.5 g, 0.013 mol) were dissolved in tetrahydrofuran(10 mL). Then lump Na⁰ (0.030 g, 0.013 mol) was added and the reactionwas stirred for 4 h. Crude reaction was quenched with 1M HCl (20 mL),washed ethyl acetate (3×20 mL). Organic layers were combined and washedwith H₂O (2×10 mL), dried MgSO₄, and evaporated to dryness to afford 2 ayellowish oil (1.07 g, 30% yield).

8.15 Synthesis of 4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy{-butyricacid

4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid ethyl ester(1.07 g, 4.0 mmol) was dissolved in 1M NaOH (10 mL) and the reaction wasstirred for 2 h. Crude reaction was diluted with sat. NaCl (40 mL),acidified to pH ˜2 with conc. HCl, washed CH₂Cl₂ (2×50 mL), dried MgSO₄,and evaporated to dryness to afford 3 a clear oil (0.945 g, 94% yield).

8.16 Synthesis of 4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyricacid 2,5-dioxo-pyrrolidin-1-yl ester

N-hydroxysuccinimide (0.55 g, 4.8 mmol) and EDCI (1.15 g, 6.0 mmol) weredissolved in CH₂Cl₂ (7 mL). Then4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid (0.940 g, 3.8mmol), dissolved in CH₂Cl₂ (2 mL), was added. Reaction stirred overnightat room temperature. Crude reaction was diluted with CH₂Cl₂ (21 mL),washed 1M HCl (30 mL), H₂O (30 mL), dried MgSO₄, and evaporated todryness. Column chromatography (Silica, ethyl acetate) afforded 4, aclear oil (0.556 g, 43% yield).

9 PREPARATION OF COMPLEXES

Methods were investigated for the preparation of zinc complexes ofinsulin compound conjugates. New methods, exceptional to publishedmethods used for complexation/crystallization of insulin compound andinsulin compound analogs, were developed to make zinc complex of HIM2.HIM2 is a human insulin monoconjugate with a modifying moiety coupled atB29, where the modifying moiety has the following structure:

Further complexes were prepared using IN105, a human insulinmonoconjugate with a modifying moiety coupled at B29, where themodifying moiety has the following structure:

The methods provided three main types, “T-type” and “R-type” and“protamine” cation complexes of insulin compound conjugate solids.

9.1 Preparation and Analysis of T-Type Solids

9.1.1 Attempted Preparation of T-Type Zn Complex of HIM2 (2 g/L)

A HIM2 solution at approximately 2 g/L was prepared having a final pH ˜3with 10% HCl. Glacial acetic acid was added to a 10 mL aliquot (20 mgprotein) of the above solution to a final concentration of 0.25M. Twenty(or forty) μL of a 2% w/w ZnCl₂ solution was added to the sample. The pHwas adjusted to 5.1 (or 5.5) with concentrated ammonium hydroxide. Thesolution stirred for 15 minutes at room temperature (or +5° C.) and thenstood for one day at room temperature (or +5° C.) to allow solidformation. No crystals or precipitation formed after allowing thereaction to stand one day at room temperature (or at +5° C.). SeeExample 2 of U.S. Pat. No. 5,504,188, entitled “Preparation of stablezinc insulin compound analog crystals.”

9.1.2 T-Type Zn Complex of HIM2 (10 g/L Concentration)

A HIM2 solution at approximately 10 g/L was prepared having a final pH˜3 with 10% HCl. Glacial acetic acid was added to a 10 mL (100 mgprotein) aliquot of the above solution to a final concentration of0.25M. Forty μL of a 10% w/w ZnCl2 solution was added to the sample. ThepH was adjusted to 5.20 with concentrated ammonium hydroxide. Thesolution was stirred for 15 minutes at +5° C. and then allowed to standfor five days at +5° C. to allow solid formation.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2000 RPM for 10 minutes. The solution was decanted andthe solid was washed with 5 mL cold DI water. This solution wascentrifuged at 2000 RPM for 10 minutes before the water was decanted andthe solids were washed with another 5 mL cold DI water. Again, thesample was centrifuged at about 2000 RPM for about 10 minutes before theH₂O was decanted. The sample was washed with 5 mL 200 proof cold EtOHand centrifuged at 2000 RPM for 10 minutes before the EtOH was decanted.The sample was dried in a lyophilizer to provide white solid.

9.1.3 T-Type Zn Complex of HIM2 (20 g/L Concentration)

A HIM2 solution at approximately 20 g/L was prepared having a final pH˜3 with 10% HCl. Glacial acetic acid was added to a 10 mL (200 mgprotein) aliquot of the above solution to a final concentration of 0.25M. Eighty μL of a 10% w/w ZnCl₂ solution was added to the sample. The pHwas adjusted to 5.37 with concentrated ammonium hydroxide. The solutionstirred for 15 minutes at +5° C. and then stood for four days at +5° C.to allow solid formation.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2600 RPM for 20 minutes. The solution was decanted andthe solid was washed with 5 mL cold DI water. This solution wascentrifuged at 2600 RPM for 20 minutes before the water was decanted andthe solids were washed with another 5 mL cold DI water. Again, thesample was centrifuged at about 2600 RPM for about 20 minutes before theH₂O was decanted. The sample was washed with 5 mL 200 proof cold EtOHand centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted.The sample was dried in a lyophilizer to provide white solid.

9.1.4 T-Type Zn Complex of of HIM2 (30 g/L Concentration)

A HIM2 solution at approximately 30 g/L is prepared having a final pH ˜3with 10% HCl. Glacial acetic acid was added to a 50 mL (1.5 g protein)aliquot of the above solution to a final concentration of 0.25 M. Sixhundred μL of a 10% w/w ZnCl₂ solution was added to the sample. The pHwas adjusted to 5.34 with concentrated ammonium hydroxide. The solutionstood at +5° C. for five days to allow solid formation.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2800 RPM for 15 minutes. The solution was decanted andthe solid was washed three times with 10 mL cold DI water, centrifugingand decanting the H₂O each wash. The sample was then washed three timeswith 10 mL 200 proof cold EtOH. It was centrifuged at 2800 RPM for 15minutes and decanted after each wash. The sample was dried in alyophilizer to provide white solid.

FIGS. 1 and 2 are photomicrographs taken using a Zeiss Axiovertmicroscope showing crystals grown for 24 hours. In FIG. 1, the crystalsize is approximately 11.3 μM in length and approximately 5.3 μM indiameter. In FIG. 2, the size of the crystal on the left isapproximately 15.1 μM in length and approximately 5.9 μM in diameter,and the size of the crystal on the right is approximately 9.1 μM inlength and approximately 5.3 μM in diameter. FIG. 3 is a photomicrographtaken using a Zeiss Axiovert microscope showing crystals grown for 5days. In one aspect, the invention includes crystals having a morphologyas shown in FIG. 1, 2 or 3.

9.1.5 T-Type Zn Complex of of HIM2 (50 g/L Concentration)

A HIM2 solution at approximately 50 g/L was prepared to a final pH ˜3with 10% HCl. Glacial acetic acid was added to a 10 mL aliquot of theabove solution to a final concentration of 0.25 M. Two hundred μL of a10% ZnCl₂ solution was added to the sample. The pH was adjusted to 5.23with concentrated ammonium hydroxide. The solution was stirred at +5° C.for 15 minutes and then stood at +5° C. for four days to allow solidformation to occur.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2600 RPM for 20 minutes. The solution was decanted andthe solid was washed with 5 mL cold DI H₂O. This solution wascentrifuged at 2600 RPM for 20 minutes before the H₂O was decanted andthe solid was washed with another 5 mL cold DI H₂O. Again, the samplewas centrifuged at 2600 RPM for 20 minutes before the H₂O was decanted.The sample was washed with 5 mL 200 proof cold EtOH and centrifuged at2600 RPM for 20 minutes before the EtOH was decanted. The sample wasdried in a lyophilizer for three days.

9.1.6 T-Type Zn Complex of of HIM2 (1 g Scale)

A HIM2 solution at approximately 10 g/L was prepared to a final pH ˜3with 10% HCl. Glacial acetic acid was added to a 50 mL (500 mg protein)aliquot of the above solution to a final concentration of 0.25 M. Twohundred μL of a 10% ZnCl₂ solution was added to the sample. The pH wasadjusted to 5.49 with concentrated ammonium hydroxide. The solution wasstirred at +5° C. for 15 minutes and then stood at +5° C. for seven daysto allow solid formation to occur.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2600 RPM for 20 minutes. The solution was decanted andthe solid was washed with 10 mL cold DI H₂O. This solution wascentrifuged at 2600 RPM for 20 minutes before the H₂O was decanted. Thewater washes were repeated two additional times. The sample was thenwashed with 10 mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20minutes before the EtOH was decanted. Two more EtOH washes were carriedout the same way before the sample was placed on the lyophilizer to dryfor four days.

9.1.7 T-Type Zn Complex of of HIM2 at Neutral pH (5 g Scale)

A HIM2 solution at approximately 10 g/L was prepared to a final pH ˜3with 10% HCl. Two milliliters of a 10% ZnCl₂ solution was added to thesample. The pH was adjusted to 7.05 with concentrated ammoniumhydroxide. The solution was stirred at room temperature overnight toallow solid formation to occur.

The milky Zn-HIM2 reaction mixture (500 mL) was added, in parts, to a350 mL fine-fritted (4.5-5 um) disc funnel (ChemGlass CG1402-28, 90 mmdiameter). The filtrate was collected in a side-arm flask while applyingvacuum for about 4-6 hours. As an option, the cake may be washed with100 mL cold 1% ZnCl₂ and the filtrate collected separately. The cake waswashed with 100 mL ice-cold water, and the filtrate was again collected.The cake was also washed with an additional 100 mL ice-cold 100% ethanoland the filtrate was collected once again. The final wash of the cakewas 100 mL of fresh ice-cold water and the final filtrate collected. Thecake was dried under vacuum and/or air-dried over 12-18 hours. Afterdrying, the cake was scraped off the funnel, weighed, andmoisture/protein contents were measured via HPLC. The collectedfiltrates from the various wash steps were also analyzed using HPLC todetermine the concentration of the lost Zn-HIM2 during the process. Thefiltration yielded a 2.5% w/w Zn content with an overall yield of 98%.

9.1.8 T-Type Zn Complex of HIM2 at Neutral pH (500 mg Scale)

A HIM2 solution at approximately 10 g/L was prepared to a final pH ˜3with 10% HCl. Two hundred μL of a 10% ZnCl₂ solution was added to thesample. The pH was adjusted to 7.06 with concentrated ammoniumhydroxide. The solution was stirred at +5 for 15 minutes and then stoodat +5 for two days to allow solid formation to occur.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2800 RPM for 15 minutes. The solution was decanted andthe solid was washed with 10 mL cold DI H₂O. This solution wascentrifuged at 2800 RPM for 15 minutes before the H₂O was decanted. Thewater washes were repeated two additional times. The sample was thenwashed with 10 mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20minutes before the EtOH was decanted. Two more EtOH washes were carriedout the same way before the sample was placed on the lyophilizer to dryfor two days.

9.1.9

Results for T-type solid compositions Solubility Reaction Observations(mg/mL) % w/w Zn 9.1.1 No solid formed N/A N/A 9.1.2 White solid NEM NEM9.1.3 White solid NEM NEM 9.1.4 White solid NEM 0.53 9.1.5 White solid146 0.66 9.1.6 White solid 109 0.55 9.1.7 White solid ND 2.50 9.1.8White solid ND 1.63 NEM = Not enough material ND = No data

9.2 Preparation and Analysis of R-Type Solids

9.2.1 R-Type Zn Complex of HIM2 with Phenol at 2 g/L

A HIM2 solution at approximately 2 g/L was prepared to a final pH ˜3with glacial acetic acid. Thirty three microliters of liquefied phenolwas added to a 10 mL aliquot of the above solution. The pH was adjustedto 5.89 with concentrated ammonium hydroxide. One hundred sixty μL of a10% w/w ZnCl₂ solution was added to the sample. The solution was stirredat room temperature for 15 minutes and then stood at room temperaturefor three days to allow more precipitate to form.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 3400 RPM for 15 minutes. The supernatant was decanter andthe solid was washed with 5 mL cold DI water. This solution wascentrifuged at 3200 RPM for 15 minutes before the H₂O was decanted. Thesample was then washed with 5 mL 200 proof cold EtOH and centrifuged at3200 RPM for 15 minutes before the EtOH was decanted. Again the samplewas washed with 5 mL of cold EtOH, however, it was not centrifuged. Thesolid was allowed to settle to the bottom of the tube and then placed inthe speed vacuum to dry.

9.2.2 Preparation of R-Type Zn Complex of HIM2 at 20 g/L

A HIM2 solution at approximately 20 g/L was prepared to a final pH ˜3with 10% HCl. Sixty six μL of liquefied phenol was added to a 10 mLaliquot of the above solution. The pH was adjusted to 6.43 withconcentrated ammonium hydroxide. Three hundred twenty μL of a 10% ZnCl₂solution was added to the sample. The solution was stirred at roomtemperature for 15 minutes and then stood at room temperature for fourdays to allow more precipitate to form.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2600 RPM for 20 minutes. The solution was decanted andthe solid was washed with 5 mL cold DI H₂O. This solution wascentrifuged at 2600 RPM for 20 minutes before the H₂O was decanted andthe solid was washed with another 5 mL cold DI H₂O. Again, the samplewas centrifuged at 2600 RPM for 20 minutes before the H₂O was decanted.The sample was washed with 5 mL 200 proof cold EtOH and centrifuged at2600 RPM for 20 minutes before the EtOH was decanted. The sample waslyophilized for three days.

9.2.3 Preparation of R-Type Zn Complex of HIM2 at 30 g/L

A HIM2 solution at approximately 30 g/L was prepared to a final pH ˜3with 10% HCl. Ninety nine microliters of liquefied phenol was added to a10 mL aliquot of the above solution. The pH was adjusted to 6.47 withconcentrated ammonium hydroxide. Then, 480 μL of a 10% ZnCl₂ solutionwas added to the sample. The solution was stirred at room temperaturefor 15 minutes and then stood at room temperature for four days to allowmore precipitate to form.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2600 RPM for 20 minutes. The solution was decanted andthe solid was washed with 5 mL cold DI H₂O. This solution wascentrifuged at 2600 RPM for 20 minutes before the H₂O was decanted andthe solid was washed with another 5 mL cold DI H₂O. Again, the samplewas centrifuged at 2600 RPM for 20 minutes before the H₂O was decanted.The sample was washed with 5 mL 200 proof cold EtOH and centrifuged at2600 RPM for 20 minutes before the EtOH was decanted. The sample waslyophilized for three days.

FIG. 4 shows solid grown for 4 days. The picture was taken using a ZeissAxiovert microscope. The average length of the crystals is approximately9.7 μM.

9.2.4 Preparation of R-Type Zn Complex of HIM2 at 50 g/L

A HIM2 solution at approximately 50 g/L was prepared to a final pH ˜3with 10% HCl. One hundred sixty five microliters of liquefied phenol wasadded to a 10 mL aliquot of the above solution. The pH was adjusted to6.82 with concentrated ammonium hydroxide. Eight hundred μL of a 10%ZnCl₂ solution was added to the sample. The solution was stirred at roomtemperature for 15 minutes and then stood at room temperature for fourdays to allow more precipitate to form.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2600 RPM for 20 minutes. The solution was decanted, andthe solid was washed with 5 mL cold DI H₂O. This solution wascentrifuged at 2600 RPM for 20 minutes before the H₂O was decanted andthe solid was washed with another 5 mL cold DI H₂O. Again, the samplewas centrifuged at 2600 RPM for 20 minutes before the H₂O was decanted.The sample was washed with 5 mL 200 proof cold EtOH and centrifuged at2600 RPM for 20 minutes before the EtOH was decanted. The sample waslyophilized for three days.

9.2.5 Preparation of R-Type Zn Complex of HIM2 at 1 g Scale

A HIM2 solution at approximately 10 g/L was prepared to a final pH ˜3with 10% HCl. One hundred sixty five microliters of liquefied phenol wasadded to a 50 mL aliquot of the above solution. The pH was adjusted to6.42 with concentrated ammonium hydroxide. Eight hundred μL of a 10%ZnCl₂ solution was added to the sample. The solution was stirred at roomtemperature for 15 minutes and then stood at room temperature for sevendays to allow more precipitate to form.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2600 RPM for 20 minutes. The solution was decanted andthe solid was washed with 10 mL cold DI H₂O. This solution wascentrifuged at 2600 RPM for 20 minutes before the H₂O was decanted. Twomore water washes occurred the same way. The sample was then washed with10 mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutesbefore the EtOH was decanted. Two more EtOH washes were carried out thesame way before the sample was placed on the lyophilizer for four days.

9.2.6 R-Type Zn Complex of HIM2 at 5 g Scale

A HIM2 solution at approximately 10 g/L was prepared to a final pH ˜3with 10% HCl. Fifteen hundred microliters of liquefied phenol was addedto 450 mL of the above solution. The pH was adjusted to 7.1 withconcentrated ammonium hydroxide. Eighteen hundred μL of a 10% ZnCl2solution was added to the sample. The solution was stirred at roomtemperature for 15 minutes and then stood at room temperature overnightto allow more precipitate to form.

The reaction performed above was split into three filtration trials. Intrial one, the reaction mixture was filtered through a fine frittedfunnel and then washed with a 1% ZnCl2 solution. The material was driedovernight via vacuum filtration. The second trial was filtered over amedium fitted filter which also contained filter paper. The substancewas then washed with ethanol and water and dried overnight via vacuumfiltration. Finally, the third trial was filtered through a fine fittedfunnel, washed with a 1% ZnCl2 solution and also washed with ethanol andwater. This material was also dried overnight under vacuum filtration.

Trial 1 (fine-frit, Trial 2 (filter paper, ZnCl2 wash, not medium frit,Trial 3 (fine-frit, H₂0/EtOH wash EtOH/H₂0 wash) EtOH/H₂O washes) Yield74% 93%   58% w/w % Zn 1.99  2.83 2.06% w/w % 0.033 0.45 1.28 Phenol

9.2.7 R-Type Zn Complex of HIM2 at Neutral pH

A HIM2 solution at approximately 10 g/L was prepared to a final pH ˜3with 10% HCl. One hundred sixty five microliters of liquefied phenol wasadded to 50 mL of the above solution. Then, two hundred microliters of a10% ZnCl₂ solution was added to the sample. The pH was adjusted to 7.18with concentrated ammonium hydroxide. The solution sat at roomtemperature for two days to allow precipitate to form.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2800 RPM for 20 minutes. However, the material did notsettle to the bottom of the tube initially and was therefore,centrifuged for about 2 hours. The solution was decanted and the solidwas washed with 5 mL cold DI H₂O. This solution was centrifuged at 2800RPM for 60 minutes before the H₂O was decanted. The water wash wasrepeated two more times. The sample was then washed with 5 mL 200 proofcold EtOH and centrifuged at 2800 RPM for 60 minutes before the EtOH wasdecanted. Two more EtOH washes were carried out the same way. Materialwas cloudy after the third EtOH wash and was placed in the refrigeratorovernight to allow the reactions to settle more. The solvent wasdecanted and the material was placed on the lyophilizer for 2 days.

9.2.8

Results for R-type Solid Compositions Solubility Reaction Observations(mg/mL) % w/w Zn Phenol 9.2.1 White solid NEM NEM NEM 9.2.2 White solid44.75 1.21  0.097 9.2.3 White solid 50.49 1.74 0.41 9.2.4 White solid36.24 2.32 0.52 9.2.5 White solid 47.7  1.06 0.16 9.2.6 White solid NDSee above See above 9.2.7 White solid ND 1.74 1.62 NEM = Not enoughmaterial ND = No data

9.3 Preparation and Analysis of Protamine Solids

9.3.1 Preparation of T-Type Zn Complex of HIM2 with Protamine at AcidicpH

Protamine was added to a 10 g/L stock solution of HIM2 that had a finalpH ˜3 with 10% HCl. Glacial acetic acid was added to a 10 mL aliquot(100 mg protein) of the above solution to a final concentration of 0.25M. Two hundred microliters of a 10% ZnCl₂ solution was added to thesample. The pH was adjusted with concentrated ammonium hydroxide to a pH˜5. The solution was stirred at +5° C. for 15 minutes and then stood at+5° C. for two days to allow solid formation to occur.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2600 RPM for 20 minutes. The solution was decanted andthe solid was washed with 10 mL cold DI H₂O. This solution wascentrifuged at 2600 RPM for 20 minutes before the H₂O was decanted. Twomore H₂O washed occurred the same way. The sample was then washed with10 mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutesbefore the EtOH was decanted. Two more EtOH washes were carried out thesame way before the sample was placed on the lyophilizer for two days.

9.3.2 Preparation of T-Type Zn Complex of HIM2with Protamine at NeutralpH

A HIM2 solution at approximately 30 g/L was prepared to a final pH ˜3with 10% HCl. One milliliter of glacial acetic acid was added to a 50 mLaliquot (1.5 g protein) of the above solution. Six hundred microlitersof a 10% ZnCl₂ solution was added to the reaction followed by theaddition of 225 milligrams of protamine. The pH was adjusted to 6.95with concentrated ammonium hydroxide and the reaction stood for two daysat +5° C. to allow solid formation to occur.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2600 RPM for 20 minutes. The solution was decanted andthe solid was washed with 10 mL cold DI H₂O. This solution wascentrifuged at 2600 RPM for 20 minutes before the H₂O was decanted. Twomore H₂0 washed occurred the same way. The sample was then washed with10 mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutesbefore the EtOH was decanted. Two more EtOH washes were carried out thesame way before the sample was placed on the lyophilizer for three days

9.3.3 Preparation of R-Type Zn Complex of HIM2with Protamine at AcidicpH

A HIM2 solution at approximately 10 g/L was prepared to a final pH ˜3with 10% HCl. Liquified phenol (2.48 mL) was added to a 150 mL aliquot(1.5 g protein) of the above solution. The pH of the reaction wasadjusted with concentrated ammonium hydroxide to a pH ˜6.57. Twelvemicroliters of a 10% ZnCl₂ solution was added to the reaction followedby the addition of 225 milligrams of protamine. The reaction mixturestirred at room temperature for 15 minutes before it stood for two daysat room temperature to allow solid formation to occur.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2800 RPM for 15 minutes. The solution was decanted andthe solid was washed with 50 mL cold DI H₂0. This solution wascentrifuged at 2800 RPM for 15 minutes before the H₂O was decanted. Twomore H₂O washed occurred the same way. The sample was then washed with10 mL 200 proof cold EtOH and centrifuged at 2800 RPM for 15 minutesbefore the EtOH was decanted. Two more EtOH washes were carried out thesame way before the sample was placed on the lyophilizer for two days.

9.3.4 Preparation of R-Type Zn Complex of HIM2with Protamine at NeutralpH

A HIM2 solution at approximately 10 g/L was prepared to a final pH ˜3with 10% HCl. Liquefied phenol (495 mL) was added to 150 mL reaction.Then, 600 milliliters of a 10% ZnCl₂ solution was added to reactionfollowed by the addition of 75 mg protamine. The pH was adjusted withconcentrated ammonium hydroxide to a pH of 7.01. The reaction stood forthree days at room temperature to allow solid formation.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2800 RPM for 15 minutes. The solution was decanted andthe solid was washed with 50 mL cold DI H₂O. This solution wascentrifuged at 2800 RPM for 15 minutes before the H₂O was decanted. Twomore H2O washes occurred the same way. The sample was then washed with50 mL 200 Proof cold EtOH and centrifuged at 2800 RPM for 15 minutesbefore the EtOH was decanted. Two more EtOH washes were carried out thesame way before the sample was placed on the lyophilizer for two days.

9.3.5 Results for protamine solid compositions Solubility ReactionObservations (mg/mL) % w/w Zn Phenol 9.3.1 White solid ND 0.66 N/A 9.3.2White solid ND 2.47 N/A 9.3.3 White solid 36.78 1.22 9.87 9.3.4 Whitesolid NEM NEM NEM NEM = Not enough material ND = No data

9.4 Preparation and Analysis of Complexes of Insulin CompoundDiconjugates

9.4.1 T-Type Zn Complex at A1 and B29 Insulin Compound Diconjugate

An insulin compound diconjugate having a modifying moiety—C(O)(CH₂)₅(OCH₂CH₂)₇OCH₃ coupled at B29 and A1 of human insulin(DICON-1) was added to solution at approximately 10 g/L and prepared toa final pH 3.15 with 10% HCl. Glacial acetic acid was added to a 3.75 mLaliquot of the above solution to a final concentration of 0.25 M. Then15 μL of a 10% ZnCl₂ solution was added to the sample. The pH wasadjusted to 4.90 with concentrated ammonium hydroxide. The solutionstirred for 15 minutes at +5° C. and then stood for six days at +5° C.to allow solid formation (yielded a white solid).

9.4.2 R-Type Zn Complex at A1 and B29 Insulin Compound Diconjugate

DICON-1 was added to solution at approximately 10 g/L and prepared to afinal pH 3.15 with 10% HCl. About 12 μL of liquefied phenol was added toa 3.75 mL aliquot of the above solution. The pH was adjusted to 5.75with concentrated ammonium hydroxide. Sixty μL of a 10% ZnCl₂ solutionwas added to the sample. The solution was stirred at room temperaturefor 15 minutes and then stood at room temperature for six days to allowmore precipitate to form (yielded a white solid).

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2600 RPM for 20 minutes. The solution was decanted andthe solid was washed with 5 mL cold DI H₂O. This solution wascentrifuged at 2600 RPM for 20 minutes before the H₂O was decanted andthe solid was washed with another 5 mL cold DI H₂O. Again, the samplewas centrifuged at 2600 RPM for 20 minutes before the H₂O was decanted.The sample was washed with 5 mL 200 proof cold EtOH and centrifuged at2600 RPM for 20 minutes before the EtOH was decanted. The sample waslyophilized for six days.

9.4.3 Diconjugate B1, B29 (10 mg/mL)

DICON-1 was added to solution at approximately 10 g/L and 33 μL ofliquified phenol was added. The pH was adjusted to 5.34 withconcentrated ammonium hydroxide. Then 160 μL of a 10% ZnCl₂ solution wasadded to the sample. The solution stood at room temperature for twoweeks to allow solid formation to occur (yielded a white solid).

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2800 RPM for 15 minutes. The solution was decanted andthe solid was washed three times with 5 mL cold DI H₂O. The solution wascentrifuged for 15 minutes at 2800 RPM and decanted after each wash. Thesample was then washed three times with 5 mL 200 proof cold EtOH. Againthe sample was centrifuged at 2600 RPM for 15 minutes and decanted aftereach wash. The sample was lyophilized for two days.

9.4.4 Diconjugate B1, B29 (20 mg/mL)

DICON-1 was added to solution at approximately 20 g/L and 66 microlitersof liquified phenol was added. The pH was adjusted to 7.65 withconcentrated ammonium hydroxide. Then 320 μL of a 10% ZnCl₂ solution wasadded to the sample. The solution stood at room temperature for twoweeks to allow solid formation to occur (yielded a white solid).

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2800 RPM for 15 minutes. The solution was decanted andthe solid was washed three times with 5 mL cold DI H₂O. The solution wascentrifuged for 15 minutes at 2800 RPM and decanted after each wash. Thesample was then washed three times with 5 mL 200 proof cold EtOH. Againthe sample was centrifuged at 2600 RPM for 15 minutes and decanted aftereach wash. The sample was lyophilized for two days.

9.5 Preparation and Analysis of T-Type IN105 Solids

9.5.1 T-Type Zn Complex of IN105 Monoconjugate (10 g/L Concentration)

A IN105 solution at approximately 10 g/L (100 mg) was prepared having afinal pH ˜3 with 10% HCl. 50 μL of a 10% w/w ZnCl₂ solution was added tothe sample. The pH was adjusted to 7.52 with concentrated ammoniumhydroxide. The cloudy solution was stirred and then allowed to stand forfive days at room temperature to allow solid formation.

The reaction mixture was transferred to a centrifuge tube andcentrifuged at 2900 RPM for 15 minutes. The solution was decanted andthe solid was washed with 3×10 mL cold DI water. This solution wascentrifuged at 2900 RPM for 10 minutes before the water was decanted andthe solids were washed with another portion of cold DI water. The samplethen was washed with 3×10 mL 200 proof cold EtOH and centrifuged at 2900RPM for 10 minutes before the EtOH was decanted. The sample was vacuumdried to provide white solid (90 mg).

9.5.2 T-Type Zn Complex of IN105 Monoconjugate (1 g Scale)

A IN105 solution at approximately 10 g/L (1 g) was prepared to a finalpH ˜3 with 10% HCl. Five hundred μL of a 10% ZnCl₂ solution was added tothe sample. The pH was adjusted to ˜7.4 with concentrated ammoniumhydroxide. The cloudy solution was stirred for 15 minutes and thenallowed to stand at room temperature for ˜2 days before filtration.

The reaction mixture was filtered through sintered glass funnel (fine)under house vacuum. The sintered glass funnel with filtered material wasplaced under vacuum in a glass dessicator over night to result a whitefine powder (900 mg).

9.5.3 T-Type Zn Complex of IN105 Monoconjugate at Neutral pH (5 g Scale)

A IN105 solution at approximately 10 g/L (5 g, lot#Nobex040706L) wasprepared to a final pH ˜3 with 10% HCl. Two mL of a 10% ZnCl₂ solutionwas added to the sample. The pH was adjusted to ˜7.4 with concentratedammonium hydroxide. The cloudy solution was then allowed to stand atroom temperature overnight to allow solid formation before filtration.

The reaction performed above was split into 4×50 mL centrifuge tubes andinitially centrifuged at 3200 RPM for a total 2 hours. The material wasthen centrifuged at 9000 RPM for 20 minutes and stored at 5° C. overnight. The supernatant was decanted and the solid was washed with 10 mLcold DI H₂O from each tube. The tubes were inverted and centrifuged at3200 RPM for ˜1 hour before the H₂O was decanted and the solids werewashed with another 10 mL cold DI H₂O. Again, the sample was centrifugedat 3200 RPM for ˜1 hour before the H₂O was decanted. The sample waswashed with 2×10 mL 200 proof cold EtOH and centrifuged at 3200 RPM for1 hour before the EtOH was decanted. The sample was vacuum dried for twodays to give 1.64 g (lot#Nobex040730L-A) of white powder.

9.5.4 Results for T-type IN105 solid compositions Solubility (mg/mL) ina pH of about 7.4, 0.1M phosphate Reaction Observations buffer % w/w Zn9.5.1 White solid 80-85 0.0 9.5.2 White solid 10-20 1.67 9.5.3 Whitesolid ND 1.88

9.6 Preparation and Analysis of R-Type IN105 Solids

9.6.1 R-Type Zn Complex at IN105 Conjugate with Phenol at Neutral pH

A IN105 solution at approximately 10 g/L (500 mg) was prepared to afinal pH ˜3 with 10% HCl. Two hundred μL of 10% ZnCl₂ and 165 μL ofliquefied phenol was added to the above solution. The pH was adjusted to7.37 with concentrated ammonium hydroxide. The cloudy solution sat atroom temperature for 2 days to allow solid formation before filtration.

The reaction mixture was filtered through sintered glass funnel (fine)under house vacuum. The sintered glass funnel with filtered material wasplaced in under vacuum in a glass dessicator over night to result in awhite fine powder (440 mg).

9.6.2 R-Type Zn Complex of IN105 Conjugate at 5 g Scale

A IN105 solution at approximately 10 g/L (4.2 g, lot#Nobex040706L) wasprepared to a final pH ˜3 with 10% HCl. 1.5 liquefied phenol and 1.8 mLof 10% ZnCl2 solution was added to the above solution. The pH wasadjusted to ˜7.4 with concentrated ammonium hydroxide. The very cloudysolution stood at room temperature overnight to allow more precipitateto form.

The reaction performed above was split into 4×50 mL centrifuge tubes andinitially centrifuged at 3200 RPM for 2 hours. The material was thencentrifuged at 9000 RPM for 20 minutes and stored at 5° C. over night.The supernatant was decanted and the solid was washed with 10 mL cold DIH₂O from each tube. The tubes were inverted and centrifuged at 3200 RPMfor ˜1 hour before the H₂O was decanted and the solids were washed withanother 10 mL cold DI H₂O. Again, the sample was centrifuged at 3200 RPMfor ˜1 hour before the H₂O was decanted. The sample was washed with 2×10mL 200 proof cold EtOH and centrifuged at 3200 RPM for 1 hour before theEtOH was decanted. The sample was vacuum dried for 2 days to give 2.34 gof white powder.

9.6.3 Results for R-type IN105 solid compositions Solubility % w/w % w/wReaction Observations (mg/mL)* Zn Phenol 9.6.1 White solid ND 1.85 2.379.6.2 White solid 10-25 1.71 2.66 ND = No data *In a pH of about 7.4phosphate buffer

9.7 Preparation and Analysis of Protamine IN105 Solids

9.7.1 Preparation of R-Type Zn Complex of IN105 Mono Conjugate withProtamine at Acidic pH

A IN105 solution at approximately 10 g/L is prepared to a final pH ˜3with 10% HCl. Liquified phenol (248 uL) is added to a 15 mL aliquot (150mg protein) of the above solution. The pH of the reaction is adjustedwith concentrated ammonium hydroxide to a pH ˜6.50. One microliter of a10% ZnCl₂ solution is added to the reaction followed by the addition of22.5 milligrams of protamine. The reaction mixture is stirred at roomtemperature for 15 minutes before it stood for two days at roomtemperature to allow solid formation to occur.

The reaction mixture is transferred to a centrifuge tube and centrifugedat 2800 RPM for 15 minutes. The solution is decanted and the solid iswashed with 5 mL cold DI H₂O. This solution is centrifuged at 2800 RPMfor 15 minutes before the H₂O is decanted. Two more H₂O wash is occurredthe same way. The sample is then washed with 10 mL 200 proof cold EtOHand centrifuged at 2800 RPM for 15 minutes before the EtOH is decanted.Two more EtOH washes are carried out the same way before the sample isvacuum dried over two days.

9.7.2 Preparation of R-Type Zn Complex of IN105 Conjugate with Protamineat Neutral pH

A IN105 solution at approximately 10 g/L is prepared to a final pH ˜3with 10% HCl. Liquefied phenol (49.5 uL) is added to 15 mL reaction.Then, 60 microliters of a 10% ZnCl₂ solution is added to reactionfollowed by the addition of 7.5 mg protamine. The pH is adjusted withconcentrated ammonium hydroxide to a pH of 7.00. The reaction is allowedto stand for three days at room temperature to allow solid formation.

The reaction mixture is transferred to a centrifuge tube and centrifugedat 2800 RPM for 15 minutes. The solution is decanted and the solid waswashed with 5.0 mL cold DI H₂O. This solution is centrifuged at 2800 RPMfor 15 minutes before the H₂O is decanted. Two more H₂O washes areoccurred the same way. The sample is then washed with 50 mL 200 Proofcold EtOH and centrifuged at 2800 RPM for 15 minutes before the EtOH isdecanted. Two more EtOH washes are carried out the same way before thesample is vacuum dried over two days.

9.7.3 Preparation of R-Type Crystalline Zn Complex of IN105

A crude 15 mg/mL IN105 solution containing 25% organic was pH adjustedto 3.47 using 1M HCl. Solid phenol was melted in a 40-60° C. water bathand 0.218 mL was added to reaction flask. Then 0.4 mL of 4% acidifiedaqueous ZnCl₂ solution was added to reaction. The pH of the solution wasadjusted with 1M NaOH to a final pH of 6.6. While adjusting the pH, 10mL aliquots were pulled at the following pH values: 4.8, 5.0, 5.2, 5.4,5.6, 5.8, 6.0, 6.2, 6.4 and 6.6. The samples were allowed to sit withoutstirring for 24 hours. Needle-like crystals were observed under amicroscope.

9.7.4 Preparation of R-Type Crystalline Zn Complex of IN105 Containing30% Organic

A fresh 15 mg/mL solution of MPEG₃ propionyl insulin compound wasprepared in 250 mM ammonium acetate buffer and the pH was adjusted to2.81 with 1M HCl. Liquefied phenol, 0.040 mL, and 95% EtOH, 4.25 mL,were added to the solution. Then, 0.400 mL of a 4% acidified ZnCl₂solution was added to the reaction mixture. The pH of the solution wasadjusted from 3.7 to 5.4 using 50% NH₄OH and pulling lmL aliquots ateach of the following desired pH: 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2,5.4. The samples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed needle-like crystals(see FIG. 5) from the pH range 4.0 to 5.2.

9.7.5 Preparation of R-Type Crystalline Zn Complex of IN105 in 100 mMAmmonium Acetate Buffer (30, 20 and 10% EtOH)

A fresh 15 mg/mL solution of MPEG₃ propionyl insulin compound wasprepared in 100 mM ammonium acetate buffer and the pH was adjusted to2.8 with 5M HCl. Liquefied phenol, 0.040 mL, and 95% EtOH, 4.25 mL, wereadded to the solution. Then, 0.400 mL of a 4% acidified ZnCl₂ solutionwas added to the reaction mixture. The pH of the solution was adjustedfrom 2.9 to 5.6 using 5M NH₄OH and pulling 0.5 mL aliquots at each ofthe following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6. Thesamples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed needle-like crystalsfrom the pH range 4.4 to 4.8.

A fresh 15 mg/mL solution of MPEG₃ propionyl insulin compound wasprepared in 100 mM ammonium acetate buffer and the pH was adjusted to2.8 with 5M HCl. Liquefied phenol, 0.040 mL, and 95% EtOH, 2.25 mL, wereadded to the solution. Then, 0.400 mL of a 4% acidified ZnCl₂ solutionwas added to the reaction mixture. The pH of the solution was adjustedfrom 2.9 to 5.6 using 5M NH₄OH and pulling 0.5 mL aliquots at each ofthe following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6. Thesamples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed circle-like crystalsfrom the pH range 4.8 to 5.4.

A fresh 15 mg/mL solution of MPEG₃ propionyl insulin compound wasprepared in 100 mM ammonium acetate buffer and the pH was adjusted to2.8 with 5M HCl. Liquefied phenol, 0.040 mL, and 95% EtOH, 1.15 mL, wereadded to the solution. Then, 0.400 mL of a 4% acidified ZnCl₂ solutionwas added to the reaction mixture. The pH of the solution was adjustedfrom 2.8 to 5.6 using 5M NH₄OH and pulling 0.5 mL aliquots at each ofthe following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6. Thesamples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed needle-like crystalsfrom the pH range 5.0 to 5.6.

9.7.6 Preparation of R-Type Crystalline Zn Complex of IN105 in 20%Organic with 0.1 and 0.2% Phenol

A fresh 15 mg/mL solution of MPEG₃ propionyl insulin compound wasprepared in 100 mM ammonium acetate buffer and the pH was adjusted to3.0 with 5M HCl. Liquefied phenol, 0.010 mL, and 95% EtOH, 2.5 mL, wereadded to the solution. Then, 0.400 mL of a 4% acidified ZnCl₂ solutionwas added to the reaction mixture. The pH of the solution was adjustedfrom 3.2 to 5.6 using 5M NH₄OH and pulling 0.5 mL aliquots at each ofthe following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6. Thesamples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed circle-like crystalsfrom the pH range 4.4 to 5.4.

A fresh 15 mg/mL solution of MPEG₃ propionyl insulin compound wasprepared in 100 mM ammonium acetate buffer and the pH was adjusted to3.0 with 5M HCl. Liquefied phenol, 0.020 mL, and 95% EtOH, 2.5 mL, wereadded to the solution. Then, 0.400 mL of a 4% acidified ZnCl₂ solutionwas added to the reaction mixture. The pH of the solution was adjustedfrom 3.3 to 5.6 using 5M NH₄OH and pulling 0.5 mL aliquots at each ofthe following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6. Thesamples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed circle-like crystalsfrom the pH range 4.4 to 5.2.

9.7.7 Preparation of R-Type Crystalline Zn Complex of IN105 at 8.0 GramScale, pH 4.8 and Room Temperature

A fresh 15 mg/mL solution of MPEG₃ propionyl insulin compound wasprepared in 250 mM ammonium acetate buffer and the pH was adjusted to2.0 with 5M HCl. Liquefied phenol, 2.13 mL, and 95% EtOH, 225 mL, wereadded to the solution. Then, 21.3 mL of a 4% acidified ZnCl₂ solutionwas added to the reaction mixture. The pH of the solution was adjustedto 4.8 using 5M NH₄OH. The solution was allowed to sit without stirringfor 24 hours before the crystals were harvested. Needle-like crystalswere observed at the T=0 microscope picture.

The crystals were harvested by splitting the reaction mixture into 6×250mL centrifuge tubes. The tubes were spun at 10,000 RPM for 8 minutes at10° C. before the supernatant was decanted. Then, to each tube, wasadded 10 mL cold H₂O before consolidating the 6 tubes into 2 tubes. Thecentrifuge process was repeated once more with cold water and twice morewith cold EtOH. The crystals were then dried with a desktop lyophilizerfor 2 days. The procedure produced 93% yield (w/w) relative to thestarting material.

9.7.8 Preparation of R-Type Crystalline Zn Complex of IN105 at 1.5 GramScale, pH 4.8 and Room Temperature

A fresh solution of MPEG3 Propionyl Insulin compound (IN105) wasprepared by dissolving 1.52 g of solid IN105 in 100 mL of 250 mMammonium acetate pH 7.5. The solution was adjusted to pH 2.8 using 5MHCl/5M NH₄OH. Solid phenol was melted in a 40-60° C. warm water bath.400 μL of melted phenol and 42.5 mL of 95% EtOH were added to thereaction flask. Then 4 mL of 4% acidified aqueous ZnCl₂ was added to thereaction flask. The resulting solution was then adjusted to pH 4.8 using5M NH₄OH. The reaction was then allowed to sit without stirring for 48hours before crystals were harvested. Needle-like crystal formation wasobserved after 21 hours via microscope.

The crystals were harvested by splitting of the reaction slurry among4×50 mL centrifuge tubes. The tubes were spun initially at 1000 RPM for8 min. The supernatant was then decanted. The crystals in each tube werewashed with 1×5 mL aliquot of ice-cold H₂O then spun at 3000 RPM for 8min. The supernatant was then decanted. Repeated the washing/spinningprocedure with 1×5 mL aliquot of ice-cold H₂O then with 1×5 mL aliquotof ice-cold EtOH. The crystals were then dried in a vacuum dessicatorovernight. The procedure produced 73% yield (w/w) relative to thestarting material.

9.7.9 Preparation of R-Type Crystalline Zn Complex of IN105 at 1.5 GramScale, pH 4.4 and Room Temperature

A fresh solution of MPEG3 Propionyl Insulin compound (IN105) wasprepared by dissolving 1.50 g of solid IN105 in 100 mL of 250 mMammonium acetate pH 7.5. The solution was adjusted to pH 2.6 using 5MHCl. Solid phenol was melted in a 40-60° C. warm water bath. 400 μL ofmelted phenol and 42.5 mL of 95% EtOH were added to the reaction flask.Then 4 mL of 4% acidified aqueous ZnCl₂ was added to the reaction flask.The resulting solution was then adjusted to pH 4.4 using 5M NH₄OH. Thereaction was then allowed to sit without stirring for 22 hours beforecrystals were harvested. A mixture of needle-like crystal formation andprecipitate was observed after 2 hours via microscope. The reactionmixture appeared to be completely crystalline after 21 hours viamicroscope.

The crystals were harvested by transferring the reaction slurry to a1×250 mL centrifuge tube. The tube was spun initially at 10,000 RPM for8 min. The supernatant was then decanted. The crystals were washed with1×20 mL aliquot of ice-cold H₂O then spun at 10,000 RPM for 8 min. Thesupernatant was then decanted. Repeated the washing/spinning procedurewith 1×20 mL aliquot of ice-cold H₂O then with 2×20 mL aliquots ofice-cold EtOH and a final 1×20 mL aliquot of ice-cold H₂O. The crystalswere then dried in a vacuum dessicator overnight. The procedure produced67% yield (w/w) relative to the starting material.

9.7.10 Preparation of R-Type Crystalline Zn Complex of IN105 at 8.0 GramScale, pH 4.8 and Room Temperature

A fresh solution of MPEG3 Propionyl Insulin compound (IN105) wasprepared by dissolving 7.98 g of solid IN105 in 533 mL of 250 mMammonium acetate pH 7.5. The solution was adjusted to pH 2.4 using 5MHCl. Solid phenol was melted in a 40-60° C. warm water bath. 2.13 mL ofmelted phenol and 225 mL of 95% EtOH were added to the reaction flask.Then 21.3 mL of 4% acidified aqueous ZnCl₂ was added to the reactionflask. The resulting solution was then adjusted to pH 4.8 using 5MNH₄OH. The reaction was then allowed to sit without stirring for 21hours before crystals were harvested. The reaction mixture appeared tobe completely crystalline after 2 hours via microscope.

The crystals were harvested by splitting the reaction slurry among 6×250mL centrifuge tubes. The tubes were spun initially at 10,000 RPM for 8min. The supernatant was then decanted. The crystals in each tube werewashed with 1×10 mL aliquots of ice-cold H₂O then spun at 10,000 RPM for8 min. The supernatant was then decanted Repeated the washing/spinningprocedure with 1×10 mL aliquot of ice-cold H₂O then with 2×10 mLaliquots of ice-cold EtOH and a final 1×10 mL aliquot of ice-cold H₂O.The crystals were then dried in a vacuum dessicator for 2 days. Theprocedure produced 87% yield (w/w) relative to the starting material.

9.7.11 Preparation of R-Type Crystalline Zn Complex of IN105 at 10.0Gram Scale, pH 4.8 and Room Temperature

A fresh solution of MPEG3 Propionyl Insulin compound (IN105) wasprepared by dissolving 10.06 g of solid IN105 in 670 mL of 250 mMammonium acetate pH 7.5. The solution was adjusted to pH 2.6 using 5MHCl. Solid phenol was melted in a 40-60° C. warm water bath. 2.7 mL ofmelted phenol and 285 mL of 95% EtOH were added to the reaction flask.Then 27 mL of 4% acidified aqueous ZnCl₂ was added to the reactionflask. The resulting solution was then adjusted to pH 4.8 using 5MNH₄OH. The reaction was then allowed to sit without stirring for 21hours before crystals were harvested. The reaction mixture appeared tobe completely crystalline after 2.5 hours via microscope.

The crystals were harvested by splitting the reaction slurry among 6×250mL centrifuge tubes. The tubes were spun initially at 10° C., 10,000 RPMfor 8 min. The supernatant was then decanted. The crystals in each tubewere washed with 1×10 mL aliquots of ice-cold H₂O, and consolidated into2×250 mL centrifuge tubes then spun at 10° C., 10,000 RPM for 8 min. Thesupernatant was then decanted Repeated the washing/spinning procedurewith 1×30 mL aliquot of ice-cold H₂O then with 2×30 mL aliquots ofice-cold EtOH and a final 1×30 mL aliquot of ice-cold H₂O. The crystalswere then dried using a benchtop lyopholizer for 3 days. The procedureproduced 89% yield (w/w) relative to the starting material.

9.8 Preparation and Analysis of Crystalline Zn Complex of HIM2 UsingOrganic Solvent

9.8.1 Preparation of R-Type Zn Complexes of HIM2

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.95 with 5M HCl. Liquefiedphenol, 40 uL, and 95% EtOH, 3.5 mL, were added to the solution. Then,600 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.14 to 6.0 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.2, 4.4 (See FIG. 6A), 4.6, 4.8, 5.0, 5.2, 5.4 (See FIG. 6B), 5.6, 5.8,6.0. The samples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed needle-like crystals atpH 4.4. The pH range from 4.6-6.0 show large, crystalline like solids ofvarious shapes and sizes.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.95 with 5M HCl. Liquefiedphenol, 40 uL, and 95% EtOH, 3.5 mL, were added to the solution. Then,400 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.22 to 6.0 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.2, 4.4, 4.6, 4.8, 5.0, 5.2 (See FIG. 7A), 5.4, 5.6, 5.8, 6.0. Thesamples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours show crystalline-like solidsfrom pH 4.2-6.0 of various shapes and sizes.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.95 with 5M HCl. Liquefiedphenol, 40 uL, and 95% EtOH, 3.5 mL, were added to the solution. Then,200 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.19 to 6.0 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.2, 4.4, 4.6, 4.8, 5.0 (See FIG. 7B), 5.2, 5.4, 5.6, 5.8, 6.0. Thesamples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed crystalline-like solidsfrom pH 4.4-4.6 of various shapes and sizes. The pH range of 4.8-5.2show more uniform, needle-like crystals.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.95 with 5M HCl. Liquefiedphenol, 40 uL, and 95% EtOH, 2.6 mL, were added to the solution. Then,600 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.04 to 6.0 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4 (See FIG. 8A), 5.6, 5.8, 6.0. Thesamples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed flat, snowflake-likecrystals from pH 4.6-5.4.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.95 with 5M HCl. Liquefiedphenol, 40 uL, and 95% EtOH, 2.6 mL, were added to the solution. Then,400 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.05 to 6.0 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.2, 4.4, 4.6, 4.8, 5.0 (See FIG. 8B), 5.2, 5.4, 5.6, 5.8, 6.0. Thesamples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed needle-like crystals atpH5.0, crystal-like solids at pH5.2 and flat, snowflake-like crystals atpH5.4.

Rxn 6 A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.95 with 5M HCl. Liquefiedphenol, 40 uL, and 95% EtOH, 2.6 mL, were added to the solution. Then,200 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.09 to 6.0 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.2, 4.4, 4.6, 4.8 (See FIG. 9A), 5.0, 5.2, 5.4, 5.6, 5.8, 6.0. Thesamples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed needle-like crystals andcrystal like solid at pH4.8-5.6.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.76 with 5M HCl. Liquefiedphenol, 40 uL, and 95% EtOH, 4.25 mL, were added to the solution. Then,250 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 2.97 to 5.8 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sitwithout stirring for 24 hours. The microscope pictures taken after 24hours showed crystal-like precipitation from pH 4.6-5.8

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.76 with 5M HCl. Liquefiedphenol, 40 uL, and 95% EtOH, 4.25 mL, were added to the solution. Then,200 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.06 to 5.8 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.4, 4.6, 4.8, 5.0, 5.2, 5.4 (See FIG. 9B), 5.6, 5.8. The samples wereallowed to sit without stirring for 24 hours. The microscope picturestaken after 24 hours showed crystal-like precipitation from pH 4.6-5.6.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.76 with 5M HCl. Liquefiedphenol, 40 uL, and 95% EtOH, 4.25 mL, were added to the solution. Then,150 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.09 to 5.8 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sitwithout stirring for 24 hours. The microscope pictures taken after 24hours showed crystals of various sizes and shapes from pH 5.0-5.2.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.76 with 5M HCl. Liquefiedphenol, 40 uL, and 95% EtOH, 4.25 mL, were added to the solution. Then,100 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.09 to 5.8 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.4, 4.6, 4.8, 5.0 (See FIG. 10A), 5.2, 5.4 (See FIG. 10B), 5.6, 5.8.The samples were allowed to sit without stirring for 24 hours. Themicroscope pictures taken after 24 hours showed needle-like crystals atpH 5.0 and various shapes and sizes of crystalline material from pH5.2-5.6.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.76 with 5M HCl. Liquefiedphenol, 20 uL, and 95% EtOH, 4.25 mL, were added to the solution. Then,250 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.08 to 5.8 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sitwithout stirring for 24 hours. The microscope pictures taken after 24hours showed crystal-like precipitation from pH 4.8-5.8.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.76 with 5M HCl. Liquefiedphenol, 20 uL, and 95% EtOH, 4.25 mL, were added to the solution. Then,200 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.05 to 5.8 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sitwithout stirring for 24 hours. The microscope pictures taken after 24hours showed very little crystal-like solids.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.76 with 5M HCl. Liquefiedphenol, 20 uL, and 95% EtOH, 4.25 mL, were added to the solution. Then,200 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.05 to 5.8 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sitwithout stirring for 24 hours. The microscope pictures taken after 24hours showed very little crystal-like solids.

A fresh 15 mg/mL solution of HIM2 was prepared in 250 mM ammoniumacetate buffer and the pH was adjusted to 2.76 with 5M HCl. Liquefiedphenol, 20 uL, and 95% EtOH, 4.25 mL, were added to the solution. Then,100 uL of a 4% acidified ZnCl₂ solution was added to the reactionmixture. The pH of the solution was adjusted from 3.06 to 5.8 using 5MNH₄OH and pulling 500 uL aliquots at each of the following desired pH:4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sitwithout stirring for 24 hours. The microscope pictures taken after 24hours showed very little crystal-like solids.

9.9 Co-Crystallization of HIM2 and IN105 with Zinc

9.9.1 Preparation of R-Type Co-Crystallized Zn Complexes of HIM2 andIN105

9.9.2 50:50 (HIM2:IN105)

A fresh solution of HIM2 and IN105 was prepared by dissolving 37.3 mgHIM2 and 36.4 mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 2.84 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 80 μL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 3.19 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2,5.4 and 5.6. The samples were allowed to sit without stirring for 4hours. The microscope pictures taken after 4 hours show various sizesand shapes of crystals from the pH range 4.4 to 5.6.

A fresh solution of HIM2 and IN105 was prepared by dissolving 37.1 mgHIM2 and 35.9 mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 3.03 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 40 uL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 3.38 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0 (SeeFIG. 11A), 5.2 (See FIG. 11B), 5.4 and 5.6 (See FIG. 12A). The sampleswere allowed to sit without stirring for 4 hours. The microscopepictures taken after 4 hours show mostly short, needle-like crystalsfrom the pH range 4.6 to 5.6.

9.9.3 70:30 (HIM2:IN105)

A fresh solution of HIM2 and IN105 was prepared by dissolving 53.4 mgHIM2 and 23.2 mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 2.62 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 80 uL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 3.02 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2(See FIG. 12B), 5.4 and 5.6. The samples were allowed to sit withoutstirring for 1 hour. The microscope pictures taken after 1 hour showvarious sizes and shapes of crystal-like precipitation from the pH range4.4 to 5.6.

A fresh solution of HIM2 and IN105 was prepared by dissolving 53.6 mgHIM2 and 24.5 mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 2.89 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 40 uL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 3.28 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2(See FIG. 13A), 5.4 and 5.6. The samples were allowed to sit withoutstirring for 1 hour. The microscope pictures taken after 1 hour showmostly various sizes and shapes of crystal-like precipitation from thepH range 4.6 to 4.8 and many, short, needle-like crystals from the pHrange 5.0 to 5.4.

9.9.4 30:70 (HIM2:IN105)

A fresh solution of HIM2 and IN105 was prepared by dissolving 23.3 mgHIM2 and 54.7 mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 2.84 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 80 μL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 3.27 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2,5.4 and 5.6. The samples were allowed to sit without stirring for 1hour. The microscope pictures taken after 1 hour show mostly varioussizes and shapes of crystal-like precipitation from the pH range 4.4 to5.0 and few, needle-like crystals from the pH range 5.2 to 5.6.

A fresh solution of HIM2 and IN105 was prepared by dissolving 24.8 mgHIM2 and 54.9 mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 3.09 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 40 μL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 3.47 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2,5.4 and 5.6 (See FIG. 13B). The samples were allowed to sit withoutstirring for 1 hour. The microscope pictures taken after 1 hour showsmostly various circular sizes of crystal-like precipitation from the pHrange 4.4 to 5.0 and crystals various shapes and sizes from the pH range5.2 to 5.6.

9.9.5 Preparation of R-Type Co-Crystallized Zn Complexes of HIM2 andIN105

9.9.6 50:50 (HIM2:IN105)

A fresh solution of HIM2 and IN105 was prepared by dissolving 37.4 mgHIM2 and 35.9 mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 2.60 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 40 uL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 2.15 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2,5.4 and 5.6. The samples were allowed to sit without stirring for 24hours. The microscope pictures taken after 24 hours showed crystalsolids of various shapes and sizes from pH=4.6-5.6.

9.9.7 70:30 (HIM2:IN105)

A fresh solution of HIM2 and IN105 was prepared by dissolving 57.0 mgHIM2 and 24.5 mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 2.43 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 40 uL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 2.92 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6 (See FIG. 14A),4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit withoutstirring for 24 hours. The microscope pictures taken after 24 hoursshowed needle-like crystals from pH 5.0-5.2.

9.9.8 30:70 (HIM2:IN105)

A fresh solution of HIM2 and IN105 was prepared by dissolving 24.1 mgHIM2 and 53.8 mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 2.35 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 40 μL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 2.60 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0 (SeeFIG. 14B), 5.2, 5.4 and 5.6. The samples were allowed to sit withoutstirring for 24 hours. The microscope pictures taken after 24 hoursshowed needle-like crystals from pH 5.0-5.2.

9.9.9 Preparation of R-Type Co-Crystallized Zn Complexes of HIM2 andHuman Insulin

9.9.10 50:50 (HIM2:Insulin)

A fresh solution of HIM2 and Insulin was prepared by dissolving 39.2 mgHIM2 and 36.7 mg Insulin in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 2.53 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 40 uL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 2.82 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2(See FIG. 15A), 5.4 and 5.6. The samples were allowed to sit withoutstirring for 24 hours. The microscope pictures taken after 24 hoursshowed various shapes and sizes of crystal-like solid from pH 5.2 and5.4. Many tiny, needle-like crystals were observed at pH 5.6.

9.9.11 70:30 (HIM2:Insulin)

A fresh solution of HIM2 and Insulin was prepared by dissolving 56.5 mgHIM2 and 20.2 mg Insulin in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 3.23 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 40 uL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 2.82 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2,5.4 and 5.6. The samples were allowed to sit without stirring for 24hours. The microscope pictures taken after 24 hours showed variousshapes and sizes of crystal-like solid from pH 5.2 and 5.6.

9.9.12 30:70 (HIM2:Insulin)

A fresh solution of HIM2 and Insulin was prepared by dissolving 21.8 mgHIM2 and 49.2 mg Insulin in 4 mL of 250 mM ammonium acetate pH 7.5. Thesolution was adjusted to pH 3.23 using 5M HCl. Solid phenol was meltedin a 40-60° C. warm water bath. 16 μL of melted phenol and 1.75 mL of95% EtOH were added to the reaction flask. Then, 40 uL of 4% acidifiedaqueous ZnCl₂ was added to the reaction flask. The pH of the solutionwas then adjusted from 2.93 to 5.60 using 5M NH₄OH and pulling 0.500 mLaliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2,5.4 (See FIG. 15B) and 5.6. The samples were allowed to sit withoutstirring for 24 hours. The microscope pictures taken after 24 hoursshowed flat, snowflake-like crystals at pH 4.8. At pH 5.0, there was amix of needle-like and snowflake-like crystals. From pH 5.2-5.6 therewere many tiny, needle-like crystals observed.

10 AQUEOUS SOLUBILITY OF Zn COMPLEXES

Two hundred microliters of 0.1M Phosphate Buffer Saline (PBS, filtered,pH=7.4) was added to a 1 mL conical reaction vial. To this vial, a smallamount of sample was added slowly until saturation is observed.Periodically, the solution was vortexed. Upon saturation, the vial wasplaced in a small centrifuge tube and the sample was centrifuged at 2000RPM for 3 min at RT. After centrifugation, 10 μL of the sample wasremoved from the supernatant and diluted in 490 μL buffer (0.1M PBS).This diluted sample was analyzed via HPLC to determine its'concentration.

Conjugate Solubility Zn Phenol IN105 ~26 mg/mL N/A N/A ZnIN105 15mg/mL-20 mg/mL 0.44% 0.76% ZnIN105 ~24 mg/mL 0.61% 1.11% ZnIN105 15mg/mL-20 mg/mL 0.63% 1.04%

11 IN VITRO ENZYME RESISTANCE EXAMPLES FOR Zn IN105 COMPLEXES

insulin compound conjugates (IN105) were provided in 10 mM sodiumphosphate buffer (a pH of about 7.4) and their concentrations weredetermined by HPLC (the solutions are diluted with buffer so thatequimolar comparisons can be made between parent and conjugates ˜0.6mg/mL). Lyophilized chymotrypsin enzyme was resuspended in 1 mM HCl to aconcentration of ˜7.53 U/mL. A 1.53 mL aliquot of each sample was addedto sample tubes and 0.850 mL into control tubes. Samples were tested induplicate along with four control tubes per sample. Aliquots wereincubated at 37° C. in a thermomixer for 15 minutes. Then 17 μL ofchymotrypsin enzyme was added to each sample tube. Five μL of 1 mM HClwas added to each control tube Immediately following the additions, 200μL was removed from the sample and the control tubes and placed into 50μL of 1% TFA previously aliquoted out into centrifuge tubes. This sampleserves as T=0.

The sampling procedure for Insulin compound (Zn free), IN105 (Zn free)and Insulin compound (regular insulin compound) was repeated at thefollowing intervals: 0, 2, 5, 8, 12, 15, and 30 minutes. The controlprocedure was repeated at the following intervals: 0, 8, 15, 30 minutes.For T-type and R-type samples, the procedure was repeated at thefollowing intervals: 0, 5, 8, 12, 30, 40 and 60 minutes. The controlprocedure for the Zn complexes was repeated at the following intervals:0, 12, 40 and 60 minutes. Samples were stored at −20° C. until analysiscan occur via HPLC. HPLC was performed to determine percent degradationrelative to the respective T=0 minute for each digest. The natural logof the percent remaining was plotted versus time and a linear regressionrun for each digest. The half life was calculated using the equation:t_(1/2): =−0.693/slope.

Results at 0.6 mg/ml protein: Sample T half Zinc content Phenol contentInsulin compound  4.9 mins 0.0 — (zinc free) IN105 12.5 mins 0.0 — (zincfree) IN105 11.1 mins 0.0 — (zinc free) Insulin compound, USP 11.6 mins0.3 to 1% w/w — (Regular insulin compound) IN105 55.3 mins 1.85% w/w2.37% w/w (R-type zinc complex) IN105 54.8 mins 1.88% w/w — (T-type zinccomplex)

12 FORMULATION EXAMPLES 12.1 Liquid Formulation Examples

12.1.1 Buffer solution for R6 type Zn-HIM2 buffer study ComponentsAmount in 1 mL solution Dibasic Sodium Phosphate 1.88 mg Insulincomponent 3.7 mg (100 units) Glycerol 16.0 mg Phenol (or m-Cresol) 3.0mg Zinc 0.037 mg (1% w/w of insulin)* pH 7.4 to 7.8** *Adjustment of theamount of Zinc to 0.037 mg/ml per 3.7 mg/ml insulin by adding Zincchloride and to be based the zinc content in the Zinc HIM2 solid. **pHmay be adjusted with HCl 10% and/or sodium hydroxide 10%

12.1.2 Preparation of Capric Acid/Lauric Acid Formulation Oral LiquidDiluent

We transferred approximately 60% of the required sterile water volumeinto a suitable container. We added the appropriate amount (as indicatedin the table below) of tromethamine, trolamine, citric acid anhydrous,and sodium hydroxide pellets to the container and mixed well untildissolved. We adjusted the temperature to 21-25° C. (or roomtemperature) and measured the pH of the liquid. We adjusted the pH to7.7-7.9 as necessary using 1N sodium hydroxide or 1N hydrochloric acid.We then adjusted the temperature to 45-50° C. by warming on a hotplateand maintain this temperature. We then added the capric acid to the warmsolution and mixed until the capric acid was dissolved. We adjusted thetemperature to 21-25° C. (or room temperature) and measured the pH ofthe liquid. As needed, we adjusted the pH to 7.7-7.9 using 1N sodiumhydroxide or 1N hydrochloric acid. We then mixed the solution for 5minutes. We added appropriate amount of sterile water to equal 100% ofthe required volume and mixed well.

Component Percentage (% w/v) Tromethamine 4.24 Trolamine 5.22 CitricAcid Anhydrous 6.72 Sodium Hydroxide Pellets 1.88 Capric Acid 0.50Lauric Acid 0.50 Sodium Hydroxide, 1N As Needed to Adjust pH 7.7-7.9Hydrochloric Acid, 1N As Needed to Adjust pH 7.7-7.9 Sterile WaterDilute to Required Volume

IN105, HIM2 or ZnHIM2 was weighed out in amounts necessary to achieveappropriate concentration for dosing studies, e.g., 1 mg IN105 (ofprotein) was weighed out and combined with 1 mL of formulation to yielda 1 mg/mL IN105 in formulation.

12.1.3 Preparation of Oleic Acid/Capric Acid/Lauric Acid/CholateFormulation Oral Liquid Diluent

An oral liquid formulation of R-type Zn HIM2 was prepared having thecomponents shown in the following table:

Component Percentage (% w/v) Tromethamine 4.24 Trolamine 5.22 CitricAcid Anhydrous 6.72 Sodium Hydroxide Pellets 1.88 Sodium Cholate 3.00Oleic Acid 1.00 Capric Acid 0.50 Lauric Acid 0.50 Sucralose Solution,25% 0.80 Strawberry Flavor 0.40 Sodium Hydroxide, 1N As Needed to AdjustpH 7.7-7.9 Hydrochloric Acid, 1N As Needed to Adjust pH 7.7-7.9 SterileWater Dilute to Required Volume

Oral liquid samples were prepared to contain 1 mg/mL protein equivalentof R-type Zn HIM2 (ZnHIM2-R). The ZnHIM2-R was removed from the Freezer(−20° C.), placed in a dessicator and allowed to come to roomtemperature. A 1 mg/mL protein equivalent of ZnHIM2-R was prepared inoral liquid diluent solution as follows. 6.4 mg of ZnHIM2-R was weighed.Then, 5.0 mL of oral liquid diluent was transferred into the containerand gently swirled to mix. The solution took approximately 45 minutes todissolve. The resulting solution was a suspension (cloudy appearance).Prior to dosing, the solution was gently swirled for 60 seconds toensure the solution was a homogeneous solution. For ZnHIM2-R, theprotein content was 78.6%, 1 mg/mL protein equivalent, quantity=5 mL.Amount of ZnHIM2-R=(1 mg/mL)/(0.786)}×(5.0 mL)=6.4 mg. ZnHIM2-RConcentration=(6.4 mg)/(5.0 mL)=1.28 mg/mL (equivalent to 1 mg/mLadjusted for protein content).

12.1.4 Preparation of Capric Acid Liquid Formulations

We transferred approximately 60% of the required sterile water volumeinto a suitable container. We added the appropriate amount (as indicatedin the table below) of tromethamine, trolamine, citric acid anhydrous,and sodium hydroxide pellets to the container and mixed well untildissolved. We adjusted the temperature to 21-25° C. (or roomtemperature) and measured the pH of the liquid. We adjusted the pH to7.7-7.9 as necessary using 1N sodium hydroxide or 1N hydrochloric acid.We then adjusted the temperature to 45-50° C. by warming on a hotplateand maintained this temperature. We then added the capric acid to thewarm solution and mixed until the capric acid was dissolved. We adjustedthe temperature to 21-25° C. (or room temperature) and measured the pHof the liquid. As needed, we adjusted the pH to 7.7-7.9 using 1N sodiumhydroxide or 1N hydrochloric acid. We then mixed the solution for 5minutes. We added appropriate amount of sterile water to equal 100% ofthe required volume and mixed well.

Component % w/v Tromethamine 4.24 Trolamine 5.22 Citric Acid Anhydrous6.72 Sodium Hydroxide Pellets 1.88 Capric Acid 0.9, 1.5, 3.0 or 6.0Sodium Hydroxide, 1N As Needed to Adjust pH Hydrochloric Acid, 1N AsNeeded to Adjust pH Sterile Water To Required Volume

IN105 was weighed out in amounts necessary to achieve appropriateconcentration for dosing studies, e.g., 1 mg IN105 (of protein) wasweighed out and combined with 1 mL of formulation to yield a 1 mg/mLIN105 in formulation.

12.1.5 Caprate and/or Laurate in Phosphate Buffer Liquid Formulations

Preparation of 100 mM Sodium Phosphate Buffer, pH 7.8 or 8.2. Wetransferred 1.17 grams of Monosodium Phosphate Monohydrate to a 1-Lflask. Approximately 500 mL of sterile water was added and mixed welluntil dissolved. We then added 24.58 grams of Sodium Phosphate DibasicHeptahydrate and mixed well until dissolved. Diluted to volume withsterile water and mixed well. Filtered through a 0.22 μm filter. Weadjusted the pH to 7.8 or 8.2 with 1N HCl or 1N NaOH.

For IN105, we transferred 60% of the appropriate volume of the phosphatebuffer pH 7.8 or 8.2 into a suitable container. We then added an amountof caprate calculated to produce 3% w/v of the final solution and mixedwell until dissolved. We then adjusted the pH to 7.8 or 8.2 with 1N HClor 1N NaOH. We diluted to the appropriate volume (e.g., 100 mL) withphosphate buffer pH 7.8 or 8.2.

Component % w/v Sodium Caprate 3.0 100 mM Sodium Phosphate Buffer, pH7.8 or 8.2 QS to 100%

For BN-054, we weighed 400 grams of 100 mM Sodium Phosphate Buffer, pH7.8 in a suitable container. We added 9.7 grams of Sodium Caprate and11.1 grams Sodium Laurate and mixed well until dissolved. We addedappropriate amount of 100 mM Sodium Phosphate Buffer, pH 7.8, to equal anet weight of 500 grams.

Component % w/w Sodium Caprate 1.94 Sodium Laurate 2.22 100 mM SodiumPhosphate Buffer, pH 7.8 QS to 100%

12.1.6 Liquid Formulation with Arginine or Trolamine

Preparation of 100 mM Sodium Phosphate Buffer, pH 7.8. We transferred1.17 grams of Monosodium Phosphate Monohydrate to a 1-L flask.Approximately 500 mL of sterile water was added and mixed well untildissolved. We then added 24.58 grams of Sodium Phosphate DibasicHeptahydrate and mixed well until dissolved. Diluted to volume withsterile water and mixed well. Filtered through a 0.22 μm filter. Weadjusted the pH to 7.8 with 1N HCl or 1N NaOH.

We transferred 60% of the appropriate volume of the phosphate buffer pH7.8 into a suitable container. We added the appropriate amount (asindicated in the table below) of arginine or trolamine to the containerand mixed well until dissolved. We then added an amount of capratecalculated to produce 3% w/v of the final solution and mixed well untildissolved. We then adjusted the pH to 7.8 with 1N HCl or 1N NaOH. Wediluted to the appropriate volume (e.g., 100 mL) with phosphate bufferpH 7.8.

Component % w/v Sodium Caprate 3.0 Arginine or Trolamine 0.4 or 1.2 100mM Sodium Phosphate Buffer, pH 7.8 QS to 100%

IN105 was weighed out in amounts necessary to achieve appropriateconcentration for dosing studies, e.g., 1 mg IN105 (of protein) wasweighed out and combined with 1 mL of formulation to yield a 1 mg/mLIN105 in formulation.

12.1.7 Liquid Formulation with Caprylic Acid

We transferred approximately 60% of the required sterile water volumeinto a suitable container. We added the appropriate amount (as indicatedin the table below) of tromethamine, trolamine, citric acid anhydrous,and sodium hydroxide pellets to the container and mixed well untildissolved. We adjusted the temperature to 21-25° C. (or roomtemperature) and measured the pH of the liquid. We adjusted the pH to7.7-7.9 as necessary using 1N sodium hydroxide or 1N hydrochloric acid.We then adjusted the temperature to 45-50° C. by warming on a hotplateand maintained this temperature. We then added the caprylic acid to thewarm solution and mixed until the caprylic acid is dissolved. Weadjusted the temperature to 21-25° C. (or room temperature) and measuredthe pH of the liquid. As needed, we adjusted the pH to 7.7-7.9 using 1Nsodium hydroxide or 1N hydrochloric acid. We then mixed the solution for5 minutes. We added appropriate amount of sterile water to equal 100% ofthe required volume and mixed well.

Component % w/v Tromethamine 4.24 Trolamine 5.22 Citric Acid Anhydrous6.72 Sodium Hydroxide Pellets 1.88 Caprylic Acid 3.0  Sodium Hydroxide,1N As Needed to Adjust pH Hydrochloric Acid, 1N As Needed to Adjust pHSterile Water To Required Volume

IN105 was weighed out in amounts necessary to achieve appropriateconcentration for dosing studies, e.g., 1 mg IN105 (of protein) wasweighed out and combined with 1 mL of formulation to yield a 1 mg/mLIN105 in formulation.

12.1.8 Liquid Formulation with Linoleic Acid

Preparation of 100 mM Sodium Phosphate Buffer, pH 7.8. We transferred1.17 grams of Monosodium Phosphate Monohydrate to a 1-L flask.Approximately 500 mL of sterile water was added and mixed well untildissolved. We then added 24.58 grams of Sodium Phosphate DibasicHeptahydrate and mixed well until dissolved. Diluted to volume withsterile water and mixed well. Filtered through a 0.22 μm filter. Weadjusted the pH to 7.8 with 1N HCl or 1N NaOH.

We transferred 60% of the appropriate volume of the phosphate buffer pH7.8 into a suitable container. We then added an amount of linoleic acidsodium salt calculated to produce 3% w/v of the final solution and mixedwell until dissolved. We then adjusted the pH to 7.8 with 1N HCl or 1NNaOH. We diluted to the appropriate volume (e.g., 100 mL) with phosphatebuffer pH 7.8.

Component % w/v Linoleic Acid Sodium Salt 3.0 100 mM Sodium PhosphateBuffer, pH 7.8 QS to 100%

12.1.9 Preparation of Capric Acid and Lauric Acid Liquid Formulations

We transferred approximately 60% of the required sterile water volumeinto a suitable container. We added the appropriate amount (as indicatedin the table below) of tromethamine, trolamine, citric acid anhydrous,and sodium hydroxide pellets to the container and mixed well untildissolved. We adjusted the temperature to 21-25° C. (or roomtemperature) and measured the pH of the liquid. We adjusted the pH to7.7-7.9 as necessary using 1N sodium hydroxide or 1N hydrochloric acid.We then adjusted the temperature to 45-50° C. by warming on a hotplateand maintained this temperature. We then added the capric acid andlauric acid to the warm solution and mixed until the capric acid andlauric acid were dissolved. We adjusted the temperature to 21-25° C. (orroom temperature) and measured the pH of the liquid. As needed, weadjusted the pH to 7.7-7.9 using 1N sodium hydroxide or 1N hydrochloricacid. We then mixed the solution for 5 minutes. We added appropriateamount of sterile water to equal 100% of the required volume and mixedwell.

Component % w/v Tromethamine 4.24 Trolamine 5.22 Citric Acid Anhydrous6.72 Sodium Hydroxide Pellets 1.88 Capric Acid 0, 0.1 or 0.9 Lauric Acid0, 0.9 or 0.1 Sodium Hydroxide, 1N As Needed to Adjust pH HydrochloricAcid, 1N As Needed to Adjust pH Sterile Water To Required Volume

IN105 was weighed out in amounts necessary to achieve appropriateconcentration for dosing studies, e.g., 1 mg IN105 (of protein) wasweighed out and combined with 1 mL of formulation to yield a 1 mg/mLIN105 in formulation.

12.2 Solid Formulation Examples

12.2.1 Preparation and Dissolution Profile of Caprate/Laurate SolidFormulation Using Nobex-IN105-[854]-

Transfer approximately 58 mg of sodium caprate, 57 mg of sodium laurate,286 mg mannitol, 30 mg of croscarmellose sodium, and 6 mg (protein) ofNobex-IN105 onto a piece of weigh paper and blend thoroughly. Transferthe blend to the press and compress at approximately 350 psi to form atablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105-[854] 58 mg Caprateand 57 mg Laurate per Tablet

Component mg per Tablet Sodium Caprate 58 Sodium Laurate 57 Mannitol 286Explotab (Croscarmellose Sodium) 30 Nobex-IN105 (protein) 6

The dissolution testing was carried out using a USP apparatus 2dissolution unit. The medium was water, paddle speed 50 rpm, and themedium volume was 500 mL. The dissolution samples were analyzed by HPLCusing a gradient system. The mobile phases were water with 0.1% TFA(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). Thegradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65%mobile phase A, 15 minutes 20% mobile phase A, 16 minutes 20% mobilephase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm andcolumn was a C18 (150×2 mm) The following tables and graphs summarizethe dissolution data obtained for the dissolution testing ofNobex-Zn-IN105 Tablets Formulation [854] containing 6 mg Zn-IN105(protein), 286 mg Mannitol, 58 mg Sodium Caprate, 57 mg Sodium Laurate,and 30 mg Croscarmellose Sodium (Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[854], % IN105 Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes)(% Dissolved) (% Dissolved) (% Dissolved) 5 71 72 72 10 86 94 90 15 8796 92 30 89 96 93 45 90 96 93 60 87 96 92

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[854], % Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average(Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 98 93 96 10 102104 103 15 101 104 103 30 102 105 104 45 102 104 103 60 102 105 104

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[854], % Laurate Dissolved Sample Time Vessel 1 Vessel 2 Average(Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 72 75 74 10 90 8990 15 91 90 91 30 88 91 90 45 89 91 90 60 91 90 91

12.2.2 Solid Dosage Form (Tablet) Formulation Preparation 143 mg Caprateand 140 mg Laurate per Tablet

Preparation of Formulation Nobex-IN105-[856]-

Transfer approximately 143 mg of sodium caprate, 140 mg of sodiumlaurate, 150 mg mannitol, 30 mg of croscarmellose sodium, and 6 mg(protein) of Nobex-IN105 onto a piece of weigh paper and blendthoroughly. Transfer the blend to the press and compress atapproximately 350 psi to form a tablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105-[856] 143 mg Caprateand 140 mg Laurate per Tablet Component mg per Tablet Sodium Caprate 143Sodium Laurate 140 Mannitol 150 Explotab (Croscarmellose Sodium) 30Nobex-IN105 (protein) 6

The dissolution testing was carried out using a USP apparatus 2dissolution unit. The medium was water, paddle speed 50 rpm, and themedium volume was 500 mL. The dissolution samples were analyzed by HPLCusing a gradient system. The mobile phases were water with 0.1% TFA(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). Thegradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65%mobile phase A, 15 minutes 20% mobile phase A, 16 minutes 20% mobilephase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm andcolumn was a C18 (150×2 mm) The following tables and graphs summarizethe dissolution data obtained for the dissolution testing ofNobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mgMannitol, 143 mg Sodium Caprate, 140 mg Sodium Laurate, and 30 mgCroscarmellose Sodium (Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[856], % IN105 Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes)(% Dissolved) (% Dissolved) (% Dissolved) 5 43 31 37 10 66 53 60 15 8172 77 30 98 97 98 45 98 99 99 60 96 98 97

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[856], % Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average(Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 36 32 34 10 68 5763 15 89 79 84 30 105 103 104 45 105 103 104 60 105 104 105

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[856], % Laurate Dissolved Sample Time Vessel 1 Vessel 2 Average(Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 35 25 30 10 61 4453 15 74 61 68 30 93 88 91 45 93 91 92 60 93 92 93

12.2.3 Solid Dosage Form (Tablet) Formulation Preparation 143 mg CapratePer Tablet

Preparation of Formulation Nobex-IN105-[859]

Transfer approximately 143 mg of sodium caprate, 150 mg mannitol, 30 mgof croscarmellose sodium, and 6 mg (protein) of Nobex-IN105 onto a pieceof weigh paper and blend thoroughly. Transfer the blend to the press andcompress at approximately 350 psi to form a tablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105-[859] 143 mg Caprateper Tablet Component mg per Tablet Sodium Caprate 143 Mannitol 150Explotab (Croscarmellose Sodium) 30 Nobex-IN105 (protein) 6

The dissolution testing was carried out using a USP apparatus 2dissolution unit. The medium was water, paddle speed 50 rpm, and themedium volume was 500 mL. The dissolution samples were analyzed by HPLCusing a gradient system. The mobile phases were water with 0.1% TFA(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). Thegradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65%mobile phase A, 15 minutes 20% mobile phase A, 16 minutes 20% mobilephase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm andcolumn was a C18 (150×2 mm) The following tables and graphs summarizethe dissolution data obtained for the dissolution testing ofNobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mgMannitol, 143 mg Sodium Caprate, and 30 mg Croscarmellose Sodium(Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[859], % IN105 Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes)(% Dissolved) (% Dissolved) (% Dissolved) 5 11 26 19 10 63 58 61 15 8480 82 30 86 88 87 45 88 88 88 60 87 89 88

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[859], % Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average(Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 61 43 52 10 93 7283 15 99 95 97 30 99 100 100 45 99 100 100 60 99 100 100

12.2.4 Solid Dosage Form (Tablet) Formulation Preparation 286 mg CapratePer Tablet

Preparation of Formulation Nobex-IN105-[860]

Transfer approximately 286 mg of sodium caprate, 150 mg mannitol, 30 mgof croscarmellose sodium, and 6 mg (protein) of Nobex-IN105 onto a pieceof weigh paper and blend thoroughly. Transfer the blend to the press andcompress at approximately 350 psi to form a tablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105-[860] 286 mg Caprateper Tablet Component mg per Tablet Sodium Caprate 286 Mannitol 150Explotab (Croscarmellose Sodium) 30 Nobex-IN105 (protein) 6

The dissolution testing was carried out using a USP apparatus 2dissolution unit. The medium was water, paddle speed 50 rpm, and themedium volume was 500 mL. The dissolution samples were analyzed by HPLCusing a gradient system. The mobile phases were water with 0.1% TFA(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). Thegradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65%mobile phase A, 15 minutes 20% mobile phase A, 16 minutes 20% mobilephase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm andcolumn was a C18 (150×2 mm) The following tables and graphs summarizethe dissolution data obtained for the dissolution testing ofNobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mgMannitol, 286 mg Sodium Caprate, and 30 mg Croscarmellose Sodium(Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[860], % IN105 Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes)(% Dissolved) (% Dissolved) (% Dissolved) 5 28 19 24 10 53 44 49 15 7068 69 30 92 90 91 45 92 92 92 60 92 93 93

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[860], % Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average(Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 29 35 32 10 52 6659 15 70 84 77 30 99 99 99 45 99 99 99 60 99 100 100

12.2.5 Solid Dosage Form (Tablet) Formulation Preparation 100 mg CapratePer Tablet

Preparation of Formulation Nobex-IN105-[861]

Transfer approximately 100 mg of sodium caprate, 150 mg mannitol, 25 mgof croscarmellose sodium, and 6 mg (protein) of Nobex-IN105 onto a pieceof weigh paper and blend thoroughly. Transfer the blend to the press andcompress at approximately 350 psi to form a tablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105-[861] 100 mg Caprateper Tablet Component mg per Tablet Sodium Caprate 100 Mannitol 150Explotab (Croscarmellose Sodium) 25 Nobex-IN105 (protein) 6

The dissolution testing was carried out using a USP apparatus 2dissolution unit. The medium was water, paddle speed 50 rpm, and themedium volume was 500 mL. The dissolution samples were analyzed by HPLCusing a gradient system. The mobile phases were water with 0.1% TFA(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). Thegradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65%mobile phase A, 15 minutes 20% mobile phase A, 16 minutes 20% mobilephase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm andcolumn was a C18 (150×2 mm) The following tables and graphs summarizethe dissolution data obtained for the dissolution testing ofNobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mgMannitol, 100 mg Sodium Caprate, and 25 mg Croscarmellose Sodium(Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[861], % IN105 Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes)(% Dissolved) (% Dissolved) (% Dissolved) 5 77 41 59 10 94 84 89 15 9690 93 30 95 91 93 45 95 91 93 60 97 89 93

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[861], % Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average(Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 97 77 87 10 101 99100 15 101 103 102 30 101 103 102 45 101 104 103 60 101 104 103

12.2.6 Solid Dosage Form (Tablet) Formulation Preparation 286 mg CapratePer Tablet

Preparation of Formulation Nobex-IN105-[860]

Transfer approximately 286 mg of sodium caprate, 150 mg mannitol, 30 mgof croscarmellose sodium, and 6 mg (protein) of Nobex-IN105 onto a pieceof weigh paper and blend thoroughly. Transfer the blend to the press andcompress at approximately 350 psi to form a tablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105-[860] 286 mg Caprateper Tablet Component mg per Tablet Sodium Caprate 286 Mannitol 150Explotab (Croscarmellose Sodium) 30 Nobex-IN105 (protein) 6

The dissolution testing was carried out using a USP apparatus 2dissolution unit. The medium was water, paddle speed 50 rpm, and themedium volume was 500 mL. The dissolution samples were analyzed by HPLCusing a gradient system. The mobile phases were water with 0.1% TFA(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). Thegradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65%mobile phase A, 15 minutes 20% mobile phase A, 16 minutes 20% mobilephase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm andcolumn was a C18 (150×2 mm) The following tables and graphs summarizethe dissolution data obtained for the dissolution testing ofNobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mgMannitol, 286 mg Sodium Caprate, and 30 mg Croscarmellose Sodium(Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[860], % IN105 Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes)(% Dissolved) (% Dissolved) (% Dissolved) 5 28 19 24 10 53 44 49 15 7068 69 30 92 90 91 45 92 92 92 60 92 93 93

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[860], % Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average(Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 29 35 32 10 52 6659 15 70 84 77 30 99 99 99 45 99 99 99 60 99 100 100

12.2.7 Solid Dosage Form (Tablet) Formulation Preparation 150 mg CapratePer Tablet

Preparation of Formulation Nobex-IN105-[862]

Transfer approximately 150 mg of sodium caprate, 150 mg mannitol, 25 mgof croscarmellose sodium, and 6 mg (protein) of Nobex-IN105 onto a pieceof weigh paper and blend thoroughly. Transfer the blend to the press andcompress at approximately 350 psi to form a tablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105-[862] 150 mg Caprateper Tablet Component mg per Tablet Sodium Caprate 150 Mannitol 150Explotab (Croscarmellose Sodium) 25 Nobex-IN105 (protein) 6

The dissolution testing was carried out using a USP apparatus 2dissolution unit. The medium was water, paddle speed 50 rpm, and themedium volume was 500 mL. The dissolution samples were analyzed by HPLCusing a gradient system. The mobile phases were water with 0.1% TFA(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). Thegradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65%mobile phase A, 15 minutes 20% mobile phase A, 16 minutes 20% mobilephase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm andcolumn was a C18 (150×2 mm) The following tables and graphs summarizethe dissolution data obtained for the dissolution testing ofNobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mgMannitol, 150 mg Sodium Caprate, and 25 mg Croscarmellose Sodium(Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[862], % IN105 Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes)(% Dissolved) (% Dissolved) (% Dissolved) 5 76 41 59 10 93 87 90 15 9599 97 30 96 98 97 45 97 98 98 60 98 97 98

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets[862], % Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average(Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 84 51 68 10 98 8893 15 98 97 98 30 98 98 98 45 98 98 98 60 98 98 98

13 IN VITRO ENZYME RESISTANCE EXAMPLES

insulin compound conjugates (HIM2) were provided in 10 mM sodiumphosphate buffer (a pH of about 7.4) and their concentrations aredetermined by HPLC (the solutions are diluted with buffer so thatequimolar comparisons can be made between parent and conjugates ˜0.6mg/mL). Lyophilized chymotrypsin enzyme was resuspended in 1 mM HCl to aconcentration of 7.53 U/mL. A 1.53 mL aliquot of each sample was addedto sample tubes and 0.850 mL into control tubes. Samples were tested induplicate along with four control tubes per sample. Aliquots wereincubated at 37° C. in a thermomixer for 15 minutes. Then 17 μL ofchymotrypsin enzyme was added to each sample tube. Five μL of 1 mM HClwas added to each control tube Immediately following the additions, 200μL was removed from the sample and the control tubes and placed into 50μL of 1% TFA previously aliquoted out into centrifuge tubes. This sampleserves as T=0.

The sampling procedure for Insulin compound (Zn free), HIM2 (Zn free)and Insulin compound (regular insulin compound) was repeated at thefollowing intervals: 0, 2, 5, 8, 12, 15, and 30 minutes. The controlprocedure was repeated at the following intervals: 0, 8, 15, 30 minutes.For T-type and R-type samples, the procedure was repeated at thefollowing intervals: 0, 5, 8, 12, 30, 40 and 60 minutes. The controlprocedure for the Zn complexes was repeated at the following intervals:0, 12, 40 and 60 minutes. Samples were stored at −20° C. until analysiscan occur via HPLC. HPLC was performed to determine percent degradationrelative to the respective T=0 minute for each digest. The natural logof the percent remaining was plotted versus time and a linear regressionrun for each digest. The half life was calculated using the equation:t_(1/2): =−0.693/slope.

Results at 0.6 mg/ml protein Sample T half Zinc content Phenol contentInsulin compound 7-9 mins — — (zinc free) HIM2 12-15 mins — — (zincfree) Insulin compound, USP 26-29 mins 0.3 to 1% w/w — (Regular insulincompound) HIM2 51-78 mins 1.1% w/w 0.1 to 0.25% w/w (R-type zinccomplex) HIM2 51 mins 0.55% w/w — (T-type zinc complex) HIM2 95-120 mins2.0 to 2.1% w/w  5.3 to 6.2% w/w (R-type zinc/ protamine complex)DICON-1 24-26 mins ND ND (R-type zinc complex)

Results at 0.3 mg/mL protein Sample T half Zinc content Phenol contentInsulin compound 4-5 mins — — (zinc free) HIM2 7-9 mins — — (zinc free)Insulin compound, UPS 7-8 mins 0.4 to 1% w/w — (regular insulincompound) HIM2 19-21 mins 1.1% w/w 0.1 to 0.25% w/w (R-type zinccomplex) HIM2 12-15 mins 0.55% w/w — (T-type zinc complex)

14 IN VIVO EXAMPLES 14.1 Extended Mouse Blood Glucose Assay (MBGA)

Six paired-dose groups of 5 male CF-1 mice (Charles River Laboratories;25-30 g) received subcutaneous injections of either the insulin compoundconjugate (test article) or recombinant human insulin. The test articlewas reconstituted with phosphate buffer (0.01M, a pH of about 7.4)containing 0.1% w/w bovine serum albumin and dosed at 100, 66.6, 43.3,30, 20, and 13.3 μg/kg. Insulin compound was reconstituted withphosphate buffer (0.01 M, a pH of about 7.4) containing 0.1% w/w bovineserum albumin and dosed at 50, 33.3, 21.7, 15, 10, and 6.7 μg/kg. Afterreceiving a subcutaneous dose in the pocket formed by the thigh andgroin, animals were returned to their cages for 30 minutes at roomtemperature and then were quickly anesthetized and terminally bled.Blood samples were collected in heparin tubes for glucose assay. Ifglucose assay was delayed, the tubes were stored in ice water andre-warmed to room temperature before assay.

Plasma glucose was measured with a glucometer (e.g., One Touch® Basic;Lifescan), which was calibrated at the beginning of each day of useaccording to the manufacturer's instructions. The potency of the insulincompound conjugate was then calculated relative to the standard curvethat was generated for the recombinant human insulin response.Calculations were based upon the assumption that recombinant humaninsulin has a potency of 27.4 IU/mg.

Results are shown in FIGS. 16-20. FIG. 16 shows MBGA biopotency profilesfor HIM2. FIG. 17 shows MBGA biopotency profiles for Zn-HIM2 insulincompound product R-type. FIG. 18 shows MBGA biopotency profiles forZn-HIM2 insulin compound product T-type. FIG. 19 shows MBGA biopotencyprofiles for Zn-HIM2 insulin compound product with protamine. FIG. 20shows glucose lowering effect of R-type protamine complex at 30 and 90minutes post dose. These results show that the biopotency of HIM2 is notsignificantly reduced by complexation with Zn⁺⁺. The R-type protaminecomplex (see FIG. 17 [7]) shows greater glucose reduction at 30 minutesthan 90 minutes.

Further, FIGS. 21-24 show MBGA biopotency profiles for IN-186, IN-192,IN-190, IN-191, IN-189, IN-178, IN-193, IN-194, IN-185, IN-196 andIN-197, having structures as follows:

14.2 Dog Clamp Studies

14.2.1 Initial HIM2 Studies

Dogs (n=3 or 6) were prepared surgically (isoflurane anesthesia) byplacing a catheter in a femoral artery. The animals were allowed torecover for 16-17 days after which they were fasted overnight andstudied in the conscious state. After a 60 min equilibration period,there was a 20 min control period after which the drug was given bymouth. Zn⁺⁺-HIM2 R-type Insulin compound was tested in a buffersolution, prepared as shown in Example 12.1.1. In addition, R-type andNPH-type complexes were tested in oral liquid formulation that containscaprate acid and laurate acid, prepared as shown in Example 12.1.2.

All 3 test samples were tested at only one dose level (the dose levelswere identified based on the previous experimental results). The plasmaglucose level was then be clamped at a euglycemic value by infusion ofD-20 through a leg vein for 4 h. Blood samples (4 ml) were taken at −20,0, 5, 10, 20, 30, 45, 90, 120, 180 and 240 min for measurement ofglucose, insulin compound and C-peptide. Arterial blood samples wereobtained as required to clamp the plasma glucose level. A total of 72 mlof blood was taken in each experiment.

The following measurements were performed: glucose infusion rate,insulin compound concentration, C-peptide concentration, and plasmaC-peptide levels (to allow an estimation of endogenous insulin compoundrelease). The glucose infusion rate required to maintain euglycemiaprovides an index of insulin compound action.

Following the experiment the free end of the catheter was buriedsubcutaneously and the dogs were allowed to recover for two weeks priorto another study in which a different test article was used. Animalswere randomized to dose and used a total of 3 times. Total number ofdogs was 6.

FIGS. 25 and 26 show the results.

14.2.2 Initial IN105 Studies

The study was conducted on six (6) overnight fasted conscious mongreldogs which had been fed a diet of 34% protein, 46% carbohydrate, 14.5%fat and 5.5% fiber based on dry weight. Each animal had a silasticcatheter inserted into the femoral artery as described elsewhere (1)approximately three weeks prior to the experiment. On the day ofexperiment the catheter was removed from its subcutaneous pocket underlocal anesthesia. Test article: Nobex-IN105 (Lot.#KJ-173-095 &KJ-173-116) was provided in the oral fatty acid formulation(Nobex-IN-[753]-040422) at a concentration of 1.0 mg/ml. Each dogreceived 0.25 mg/kg oral dose of Nobex-IN105 (1.0 mg/ml@0.25 ml/kgdosing volume). Nobex-IN105 was given at t=0 and glucose (D-20) wasinfused through a cephalic vein in order to maintain euglycemia.Arterial blood samples were drawn for the measurement of insulin andglucose as previously described (1). After the experiment was completed,the arterial catheter was buried subcutaneously as it was during theinitial surgery.

During the experiment, one dog (Oral-2i) vomited immediately after thedosing and only a portion of the dose administered. Therefore, theresults from this experiment were reported with and without the dataobtained from this dog.

Arterial plasma insulin levels rose in all six dogs, including outlier(Oral-2i), following the oral administration of Nobex-IN105. Meanarterial insulin rose from 6.0±1.4 μU/ml (6.3±1.7 μU/ml, n=5) to a peakof 109.4±31.4 μU/ml (127.8±30.1 μU/ml, n=5) at 10 minpost-administration and then fell so that by 150 min all dogs returnedto baseline insulin levels (FIGS. 27 and 28).

Euglycemia was maintained by glucose infusion. The glucose infusion raterequired to maintain euglycemia as greatest in the animals with largerrise in arterial insulin (FIGS. 27 and 28). Mean Area-Under-The-GlucoseInfusion Rate Curve (AUCO-240) was 578.5±144.5 mg/kg/min (669.4±137.7mg/kg/min, n=5).

14.2.3 Solid Formulations

Formulation screening studies using the glucose-clamp model wereconducted on overnight fasted conscious mongrel dogs which had been feda diet of 34% protein, 46% carbohydrate, 14.5% fat and 5.5% fiber, basedon dry weight. Each animal had a silastic catheter inserted into thefemoral artery as described elsewhere (reference 1) approximately threeweeks prior to the experiment. On the day of experiment the catheter wasremoved from its subcutaneous pocket under local anesthesia. The testarticle contained 1.0 mg/mL Zn IN105 in different liquid formulations or5-6 mg of IN105 per capsule or tablet. Each dog in every experimentreceived an oral liquid dose of approximately 0.25 mg/kg or a capsule ortablet containing 5 or 6 mg of IN105 twice in succession, the first att=0 and the second at t=120 minutes. Glucose (D-20) was infused througha cephalic vein in order to maintain euglycemia. In some cases studytime was extended after dosing where the effect lasted beyond 120minutes. Arterial blood samples were drawn for the measurement ofinsulin, glucose and C-peptide as previously described (reference 1).After the experiment was completed, the arterial catheter was replacedinto the subcutaneous tissue.

Formulations, both solution and solid dosage forms, ware prepared withdifferent levels of fatty acids, buffers, diluents and disintegrants(Appendix 2: Tables 20 to 29). To minimize variables, the liquid and thesolid formulations contained consistent levels of IN105 and eachexcipient (Capric, Lauric, Caprylic, Myristic, Linoleic), thus varyingonly the relative amounts of fatty acid content, buffer, diluents(mannitol or micro crystalline cellulose), and/or disintegrant(Explotab). The glucose infusion rate and IN105 absorption (plasmainsulin immuno-reactivity) data were evaluated and compared with eachdosed formulation.

Initially, experiments were carried out to simplify and refine theoptimized liquid formulation (reference 2) to a liquid formulation thatwould be more readily converted to a solid dosage form. This was carriedout by substituting the free fatty acid with the corresponding sodiumsalt (e.g. capric acid replaced by sodium caprate) as well as removingthe buffer components (citric acid, trolamine, tromethamine, sodiumhydroxide) that were deemed to be no longer required.

Additionally, the effect of other fatty acids such as linoleic, caprylicand myristric acids and the amino acid, arginine, were examined fortheir effects on the absorption of IN105.

After an initial set of prototype screens using one to two dogs perexperiments few prototype formulations were selected and tested inadditional dogs to better determine the variability and consistencybetween formulations and individual animals.

A dissolution method was developed and dissolution studies were carriedout on a variety of candidate formulations to evaluate dissolutionprofiles of IN105 and the fatty acids contents.

Results

In the initial experiments, dogs dosed with IN105 in 3% w/v capric acidsodium salt in a phosphate buffer without additional excipients, showed(FIGS. 29-30) a similar response compared to the optimized liquidformulation containing 3% w/v capric acid in Trolamine/Citricacid/Tromethamine/Sodium Hydroxide buffer. This demonstrated that thesodium salt of the fatty acid form behaves comparably to the acid formand that the additional buffer components did not contribute to theformulation.

In separate studies evaluating alternative fatty acids substituting 3%caprate with either 3% caprylic acid or 3% linoleic acid, neither of thealternatives exhibited significant effects. The use of caprylic acidresulted in the need for low to moderate levels of glucose while the useof linoleic acid did not result in requiring any glucose infusionsuggesting lack of effect. Both formulations showed relatively lowlevels of arterial insulin. A liquid formulation containing arginineshowed relatively no benefit on GIR or IN105 levels.

In the primary studies, solid formulations were evaluated as both powderblend filled into hard gelatin capsules and tablets compressed by handusing a Carver Press. The capsules, powder blend of 6 mg of IN105(insulin equivalent, 0.25 mg/Kg) with 57 mg of caprate/57 mg laurateshowed no significant effect in the first dosing up to 120 mins, and inthe second dosing a significant effect was observed with the GIRconcurrent with IN105 absorption where the levels were well above thebase line form 0 to 120 mins (Appendix 1, Tables 10-11). This datasuggests a variable but potential delayed response in IN105 in thecapsule dosage form. Dissolution was only slightly delayed with thecapsule relative to the tablets although there may be little to noin-vitro/in-vivo correlation.

In the initial prototype tablet screening studies, tablets containing 6mg of IN105 and 150 mg Mannitol, 30 mg Exlotab with 143 mg Caparte withor with out 143 mg laurate showed significantly higher GIR and IN105absorption than the same tablets with 54 mg Caprate or/and 54 mg Laurate(FIGS. 31, 32, and 33). This suggests a reasonable dose response ofhigher GIR and IN105 levels relative to increasing levels of caprate andlaurate. The tables showed an early and consistent GIR response time tothe IN105 filled capsules. Additionally, the IN105 tablets showed anarterial plasma rise in insulin in all dogs dosed.

In a series of final studies, three prototype tablet formulations wereselected for evaluation in 5 dogs (3 different dogs for eachformulation) to assess the consistency of performance (FIGS. 34-37 and29). Arterial plasma insulin levels rose in all 3 dogs and at all 18doses (3 dogs×3 tablets×2 doses each) with corresponding GIR responsefollowing the oral administration of 6 mg (Insulin equivalent) of IN105formulated in the tablets containing either 150 mg or 280 mg caprate or140 mg/140 mg caparte/laurate (FIGS. 31 and 38-42). The c-peptide levels(ng/ml), with 150 mg and 280 mg caprate tablets, the average (n=2),showed a decrease from an initial level of 0.30±0.05 to 0.22±0.02 duringthe first dosing and 0.1±0.05 to 0.02±0.0 in the second dosing, and with140 mg/140 mg caprate/laurate tablets, showed 0.21±0.05 to 0.05±0.02during the first dosing and 0.18±0.05 to 0.18±0.01. This is indicativeof suppression of C-peptide secretion from the pancreas as result of theexogenous IN105 insulin.

All three prototype tablet formulations of IN105 showed consistentlevels of IN105 absorption and resultant glucose infusion rate amongdoses and within and between dogs, including on different days.

During the final studies, in which sets of 6 dogs were utilized, one dog(dog #3) experienced less response with all liquid and solid dosages. Tomore accurately represent the results, the data is presented with andwithout results from dog #3. Data from dogs that did not receive acomplete dose (bad gavage, vomiting, etc) or had endogenous insulin areomitted.

Dissolution study: Representative samples of tablets and capsules weresubject to dissolution testing (described above).

Discussion

These studies demonstrate that the prototype IN105 tablet containingcaprate or caprate and laurate sodium salts with mannitol and thedisintegrant Explotab and and containing 6 mg IN105 (approximately 0.25mg/kg) delivered orally resulted in significant and consistent elevationof arterial plasma insulin that required glucose infusion to preserveeuglycemia.

These prototype tablets resulted in IN105 levels and GIR rates at leastas good and likely to be better than the liquid formulations containingcomparable levels of caprate or laurate. The prototype tablets formsmaintain the absorption profile of the oral liquid formulation. Therelative oral bio-efficacy of the selected prototype tablet formulations(e.g., 280 mg and 150 mg caprate containing tablets, n=6, AUC forGIR=496±117 and 500±275) appears to be better than liquid formulations(e.g., 3% w/v capric acid liquid formulation, n=5, AUC for GIR=182±92and 198±119).

The data suggests that the tablets containing sodium caprate as the onlyfatty acid along with mannitol and the disintegrant, Explotab would beuseful in the further development of solid dosage forms for use inclinical studies. Data also suggest that Insulin levels following theoral administration of IN105 in the selected prototype caprate tabletsforms (sodium caprate at either 150 mg or 286 mg) peaked steadily with atypical Tmax at around 20 min post-dose and a C_(max) of about 59.0±20.1and 62.9.±25.4 μUnits/ml, in both doses. The plasma insulin levelsremained elevated close to the Cmax level for 10-15 minutes and abovebasal levels throughout 120 min following each dose. The GIR requiredmaintaining euglycemia using these tablets reached Tmax at or around 30to 40 min in both doses and GIR Cmax reached an average of 8.4±1.99 and7.41±2.18 mg/kg/min. The tablet dosage forms required higher GIR Cmax(7.4-8.4 vs. 4.5-5.4) and required glucose infusion for a longerduration (100-120 mins vs. 60-90 min) to maintain euglycemia then theoptimized liquid formulation.

In comparison with arterial plasma insulin levels of historical SQ andinhaled insulin, it appears that these prototype tablets provide maximuminsulin levels similar to SQ and inhaled delivery and resembles aninsulin profile comparable to that of inhaled insulin (FIGS. 34-37).

This specification is divided into sections with subject for ease ofreference only. Sections and subject headings are not intended to limitthe scope of the invention. The embodiments described herein are for thepurpose of illustrating the many aspects and attributes of the inventionand are not intended to limit the scope of the invention.

1. A solid complex comprising: an insulin compound conjugate comprisingan insulin compound conjugated to at least one modifying moiety, whereinthe insulin compound is human insulin or an analog thereof, wherein themodifying moiety comprises from 2 to 10 PEG subunits (OCH₂—CH₂) forminga PEG component coupled to a lipophilic component, where the lipophiliccomponent is selected from the group consisting of alkyls, fatty acidsand linear or branched lipids, and either the PEG component or thelipophilic component is coupled to the insulin compound on at least theB29 amino acid residue; and a cation, wherein the cation is a divalentcation selected from the group consisting of Zn⁺⁺, Mn⁺⁺, Ca⁺⁻, Fe⁺⁻,Ni⁺⁺, Cu⁺⁺, Co⁺⁺ and Mg⁺⁺; where the insulin compound conjugate iscomplexed with the cation, and wherein the solid complex is acrystalline solid or a rod shaped crystal.
 2. The complex of claim 1where the insulin compound analog is human insulin with Lys²⁸ and Pro²⁹.3. The complex of claim 1 where the insulin compound analog is a nativeproinsulin compound.
 4. The complex of claim 1 where the insulincompound analog is an artificial proinsulin compound.
 5. The complex ofclaim 1 wherein the relative lipophilicity of the insulin compoundconjugate is about 1 or less than
 1. 6. The complex of claim 1 wherein:the insulin compound is human insulin, and the relative lipophilicity isless than
 1. 7. The complex of claim 1 wherein: the cation is zinc, andaqueous solubility of the insulin compound conjugate is decreased byaddition of the zinc.
 8. The complex of claim 1 where the solubility ofthe cation-insulin compound conjugate complex at a pH of about 7.4 isless than or equal to the solubility of the insulin compound conjugate.9. The complex of claim 1 where the solubility of the cation-insulincompound conjugate complex at a pH of about 7.4 is greater than thesolubility of the insulin compound conjugate.
 10. The complex of claim 1where: the complex is an R-type complex; and at a pH of about 7.4, theaqueous solubility of the complex is from about 10 to about 150 g/L.from about 20 to about 130 g/L, from about 30 to about 110 g/L, or fromabout 35 to about 60 g/L.
 11. The complex of claim 1 where: the complexis an NPH-type complex; and at a pH of about 7.4, the aqueous solubilityof the complex is from about 1 to about 150 g/L, from about 5 to about120 g/L, or from about 10 to about 90 g/L.
 12. The complex of claim 1where: the complex is a T-type complex; and at a pH of about 7.4, theaqueous solubility of the complex is from about 30 to about 175 g/L,from about 50 to about 160 g/L, or from about 70 to about 150 g/L. 13.The complex of claim 1 where: the complex is an R-type Zn complexcomprising protamine; and at a pH of about 7.4, the aqueous solubilityof the complex is from about 10 to about 110 g/L, from about 20 to about85 g/L, or from about 30 to about 70 g/L.
 14. The complex of claim 1where: the complex is a T-type Zn complex comprising protamine; and at apH of about 7.4, the aqueous solubility of the complex is from about 10to about 150 g/L, from about 20 to about 130 g/L, from about 30 to about110 g/L, or from about 35 to about 60 g/L.
 15. The complex of claim 1where a modifying moiety is coupled at the carboxy terminus of theA-chain and/or a modifying moiety is coupled at the carboxy terminus ofthe B-chain of the insulin compound.
 16. The complex of claim 1 furthercomprising a complexing agent.
 17. The complex of claim 16 where thecomplexing agent is selected from the group consisting of protamines,surfen, globin proteins, spermine, spermidine albumin, amino acids,carboxylic acids, polycationic polymer compounds, cationic polypeptides,polylysine, anionic polypeptides, and nucleotides.
 18. A compositioncomprising the complex of claim
 1. 19. The composition of claim 18comprising two different insulin compound conjugates.
 20. Thecomposition of claim 18, where in the complex comprising a co-crystal ofat least two different insulin compound conjugates.
 21. The compositionof claim 18, wherein the complex is in the form of a crystal havingirregular morphology.
 22. The composition of claim 18, wherein thecrystal has an average diameter ranging from about 0.5 to about 40 μm.23. The composition of claim 18, wherein the complex is in the form of amixture of amorphous and crystalline solids.